BioChem Exam #2 5-8

Triacylglycerol (triglyceride)

A fat molecule consisting of an ester of glycerol with 3 fatty acids.

Eating carrots is good for both. Vitamin A, which is abundant in carrots, plats a role in vision. Diets that include generous amounts of vegetables are associated with lower incidence of cancer.

A health conscious friend asks whether eating carrots is better for the eyesight or for preventing cancer. What do you tell your friend?

Polyacrylamide

Agarose is used as a matrix for nucleic acids in electrophoresis. ________ is sued mostly for proteins.

Lewis base

An electron pair donor

K system

An enzyme for which an inhibitor or activator alters K0.5.

Not all enzyme follow Michaelis-Menten kinetics. The Kinetic behavior of allosteric enzymes does not obey the Michaelis-Menten kinetics.

Do all enzymes display kinetics that obey the Michaelis-Menten kinetics and for an allosteric enzyme?

An abzyme is created by injecting a host animal with a transition state analogue of a reaction of interest. The host animal makes antibodies to the foreign molecule, and these antibodies have specific binding points that mimic an enzyme surrounding a transition state. The purpose is to create an antibody with catalytic activity.

Explain how a researcher makes an abzyme. What is it purpose?

The number of carbons and the number of double bonds in the chain are shown by two numbers, separated by a colon. 18:1

How do you do shorthand notation for fatty acids?

The overall protein structure is needed to ensure the correct arrangement of amino acids in the active site.

If only a few of the amino acid residues of an enzyme are involved in its catalytic activity, why does the enzyme need such a large number of amino acids?

Substrate, active site

In an enzyme catalyzed reaction: _________, S: a reactant, __________ __________: the small portion of the enzyme surface where the substrate becomes bound by non-covalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions. (E + S <---> ES)

Zymogen

Inactive precursor of an enzyme where cleavage of one or more covalent bonds transforms it into the active enzyme.

Vitamin E is an antioxidant.

List an important chemical property of vitamin E.

solubility

Proteins can be purified based on _______ (usually dependent on overall charge, ionic strength, or polarity)

Feedback inhibition

The final product blocks an early reaction and shuts down the whole series.

prokaryotes

The membranes of ________, which contain no appreciable amounts of steroid, are the most fluid.

Fatty acids

Unbranched chain of 12-20 carbons with a carboxylic acid on one end. Derived from hydrolysis of animal fats, vegetable oils, or phospholipids of biological membranes.

it is stable

What does a low free energy mean?

It is the substrate concentration that leads to half of the maximal velocity. This term is used with allosteric enzymes, where the term Km is not appropriate.

What is meant by K0.5?

It is very sensitive and very accurate. Attomole (10^-18) quantities of a molecule can be detected.

What is the advantage of MALDI-TOF MS?

The cis-trans isomerization of retinal in rhodopsin triggers the transmission of an impulse to the optic nerve and is the primary photochemical event in vision.

What is the role in vision of the cis-trans isomerization of retinal?

Lock and key

____ and ____ model: substrate binds to that portion of the enzyme with a complementary shape.

general acid-base catalysis

a form of catalysis that depends on the transfer of protons

primary antibody

an antibody that will react with the protein being studied or sought during an experiment.

prostaglandins

derivatives of arachidonic acid that contain a 5 member ring and are of pharmaceutical importance.

integral proteins

proteins embedded in a membrane.

retinol

the alcohol form of vitamin A

standard free energy change

the difference between the energies of reactants and products under standard conditions

metal-ion catalysis

(Lewis acid base catalysis) a form of catalysis that depends on the Lewis definition of an acid as an electron-pair acceptor and a base as an electron-pair donor.

suicide substrates

(Trojan horse substrates) molecules used to bind to an enzyme irreversibly and inactivate it.

Waxes

A complex mixture of esters of long-chain carboxylic acids and alcohols. they are found as protective coating for plants and animals.

Glycolipids

A compound in which a carbohydrate is bound to an alcohol group (-OH) of the lipid. Many are derived from ceramides. They contain complex carbohydrate moiety that contains more than 3 sugars are known as gangliosides.

Prostaglandins

A family of compounds that have the 20-carbon skeleton of prostanoic acid. Physiologically active lipid compounds with diverse hormone-like effects in animals (i.e. vasodilation, vasoconstriction, inflammation, etc.)

Steroids

A group of lipids that have a fused ring structure of 3 six-membered rings, and 1 five membered ring. Think cholesterol.

lipids

A heterogeneous class of naturally occurring organic compounds classified together on the basis of common solubility properties.

A 100g sample of membrane contains 50 g of protein and 50 g of phosphoglycerides. 50g of lipid x (1 mol lipid/800g lipid) = 0.0625 mol lipid. 50g protein x (1 mol protein/50000g protein) = 0.001 mol protein. The molar ratio of lipid to protein is 0.0625/0.001 or 62.5/1

A membrane consists of 50% protein by weight and 50% phosphoglycerides by weight. The average molecular weight of the lipids is 800 Da. and the average molecular weight of the protein is 50,000 Da. Calculate the molar ratio of lipid to protein.

The results do not prove that the mechanism is correct because of the results from different experiments could contradict the proposed mechanisms. In that case, the mechanism would have to be modified to accommodate the new experimental results.

A model is proposed to explain the reaction catalyzed by an enzyme. Experimentally obtained rate data fit the model within experimental error. Do these findings prove the model?

Facilitated diffusion

A molecule or ion is moved across a membrane by a carrier/channel protein, down its concentration gradient.

Cofactors

A non-protein substance that takes part in an enzymatic reaction and is regenerated for further reaction.

Reversible inhibitor

A substance that binds to an enzyme to inhibit it, but can be released.

Irreversible inhibitor

A substance that causes inhibition that cannot be reversed. Usually involves formation or breaking of covalent bonds to or on the enzyme.

Allosteric effector

A substance that modifies the behavior of an allosteric enzyme; may be an : allosteric inhibitor or an allosteric activator.

Transition state analog

A substance whose shape mimics that of the transition state.

negative cooperativity

A unique feature of the sequential model is ______ _____. Induced conformational changes that make the enzyme less likely to bind to more molecules of the same type.

CTP, ATP

ATCase is an allosteric enzyme. The rate of ATCase catalysis vs substrate concentration (sigmoidal curve). What is the inhibitor? What is the activator?

Edman Degradation

After cleavage, a mixture of peptide fragment is produced. the mixture can be separated by chromatography. ____ ____ can be used to determine primary structure for the protein by looking for overlapping segments.

Heterotropic effects

Allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP.

Homotropic effects

Allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase.

It would tell you a relative concentration of the various amino acids. This is important because it would help you plan your sequencing experiment better. For example, if you had a protein whose composition showed no aromatic amino acids, it would be a waste of time to use a chymotrypsin digestion.

Amino acid compositions can be determined by heating a protein in 6 M HCl and running the hydrolysate through an ion-exchange column. If you were going to do an amino acid sequencing experiment, why would you want to get an amino acid composition first?

++Amino acids that are far apart in the amino acid sequence can be close to each other in the 3D because of protein folding. The critical amino acids are in the active site.

Amino acids that are far apart in the amino acid sequence of an enzyme can be essential for its catalytic activity. What does this suggest about its active site?

R

An allosteric activator (A) binds to concerted model and stabilizes the ____ form.

T

An allosteric inhibitor (I) binds to concerted model and stabilizes the ____ form.

Glutamic acid will be eluted first because the column pH is close to its pI. Leucine and lysine will be positively charged and will stick to the column. To elute leucine, raise the pH to around 6. To elute lysine, raise the pH to around 11.

An amino acid mixture consisting of lysine, leucine, and glutamic acid is to be separated by ion-exchange chromatography, using a cation-exchange resin at pH 3.5, with the eluting buffer at the same pH. Which of these amino acids will be eluted from the column first? Will any other treatment be needed to elute one of these amino acids from the column?

A nonpolar amino acids will stick the most to the stationary phase, so glutamic acid will move the fastest, followed by glycine and then glutamic acid.

An amino acid mixture consisting of phenylalanine, glycine, and glutamic acid is to be separated by HPLC. The stationary phase is aqueous and the mobile phase is solvent less polar than water. Which of these amino acids will move the fastest? Which one is the slowest?

Lewis acid

An electron pair acceptor

Enzymes, like all catalysts, increase the rate of the forward and reverse reactions to the same extent.

An enzyme catalyzes the formation of ATP from ADP and phosphate ion. What is its effect on the rate of hydrolysis of ATP to ADP and phosphate ion?

V system

An enzyme for which an inhibitor or activator alters Vmax but not K0.5

Yes, there would be a preference. Because the coenzyme and the other substrate will be locked into the enzyme, the hydride ion would come from some functional group that had a fixed position. Therefore, the hydride would come from one side.

An enzyme uses NAD+ as a coenzyme. Predict whether a radiolabeled H:- ion would tend to appear preferentially on one side of the nicotinamide ring as opposed to the other side.

The results do not prove that the mechanism is correct, because results from different experiments could contradict the proposed mechanism. In that case, the mechanism would have to be modified to accommodate the new experimental results.

An experiment is performed to test a suggested mechanism for an enzyme-catalyzed reaction. The results fir the model exactly (to within experimental error). Do the results prove that the mechanism is correct? Why or why not?

Instead of a phenylaline moiety (similar to the usual substrates of chymotrypsin), use a nitrogen-containing basic group similar to the usual substrates of trypsin.

An inhibitor that specifically labels chymotrypsin at histidine 57 is N-tosylamido-L-phenylethyl chloromethyl ketone. How would you modify the structure of this inhibitor to label the active site of trypsin?

Allosteric enzyme

An oligomer whose biological activity is affected by other substances binding to it. These substances change the enzyme's activity by altering the conformation of its quaternary structure.

Simple diffusion

An uncharged molecule or ion moves through the membrane, down its concentration gradient.

Most enzymes are proteins, but some catalytic RNAs (ribozymes) are known.

Are all enzymes proteins?

The relative amounts of cholesterol and phosphatidylcholine can vary widely in different types of membranes in the same cell.

Are the relative amounts of cholesterol and phophatidylcholine the same in all the kinds of membranes found in a typical mammalian cell?

feedback, CTP

Aspartate transcarbamoylase (ATCase) is regulated by _______. ATCase catalyzes the 1st step in a series of reactions that end in the production of ______(inhibitor), an important component of nucleic acids.

Temperature

Besides a metal surface or a biological catalysis, what can also catalyze reactions?

Non-competitive inhibitor

Binds to a site other than the active site; inhibits the enzyme by changing its conformation.

Competitive inhibitor

Binds to the active (catalytic) site and blocks access to it by substrate.

Vinit = (Vmax[S]/Km + [S])

By continuing to reorganize the rate equations and constants, Michaelis and Menten derived an overall equation that defines the reaction velocity in terms of the substrate concentration, the Km and the Vmax (the maximal velocity). What is the Michaelis-Menten equation?

Competitive inhibition can be overcome by adding enough substrate, but this is not true for all forms of enzyme inhibition.

Can enzyme inhibition be reversed in all cases?

The amount of product obtained in a reaction depends on the equilibrium constant. A catalyst does not affect that.

Can the presence of a catalyst increase the amount of product obtained in a reaction?

No, peroxisomes and mitochondria have overlapping sedimentation characteristics. Other techniques, such as sucrose-gradient centrifugation, would have to be used to separate the two organelles.

Can you separate mitochondria from peroxisomes using only differential centrifugation?

About 3 seconds (1 year x 1 event/10^7 events x 365 days/year x 24 hours/day x 3600 seconds/hour = 3.15 seconds).

Catalase breaks down hydrogen peroxide about 10^7 times faster than the uncatalyzed reaction. If the latter required one year, how much time would be needed by the catalase catalyzed reaction?

Absolute specificity

Catalyzes the reaction of one unique substrate to a particular product.

peptide, ester

Chymotrypsin catalyzes: the selective hydrolysis of ______ bonds where the carboxyl is contributed by Phe and Tyr. The hydrolysis of ______ bonds.

close to each other

Chymotrypsin has 3 amino acids (Ser-195, His-57, and Asp-102) required for the enzyme activity. Where are they located in the active site?

hyperbolic, sigmoidal

Chymotrypsin operates via Michaelis-Menten kinetics and exhibits a ______ curve; whereas, ATCase is an allosteric enzyme that does not operates by Michaelis-Menten kinetics and exhibits a _______ curve due to cooperativity.

Many of the double bonds have been saturated. Crisco contains "partially hydrogenated vegetable oils"

Crisco is made from vegetable oils, which are usually liquid. Why is Crisco a solid?

A glycoprotein is formed by covalent bonding between a carbohydrate and a protein, whereas a glycolipid is formed by covalent bonding between a carbohydrate and a lipid.

Define glycoprotein and glycolipid.

An omega-3 fatty acid has a double bond at the 3rd carbon from the methyl end.

Define omega-3 fatty acid.

The steady-state assumption is that the concentration of the ES complex does not change appreciably over the time in which the experiment takes place. The rate of appearance of the complex is set equal to its rate of disappearance, simplifying the equations for enzyme kinetics.

Define steady state, and comment on the relevance of this concept to theories of enzyme reactivity.

Isolate the mitochondria via differential or sucrose-gradient centrifugation. Use another homogenization technique, combined with a strong detergent, to release the enzyme from the membrane.

Describe a procedure for isolating a protein that is strongly embedded in the mitochondrial membrane.

In the first step of the reaction, the serine hydroxyl is the nucleophile that attacks the substrate peptide bond. In the second step, water is the nucleophile that attacks the acyl-enzyme intermediate.

Describe the role of nucleophilic catalysis in the mechanism of chymotrypsin.

Set up an anion-exchange column, such as Q-sepharose (quaternary amine). Run the column at pH 8.5, a pH at which the protein X has a net negative charge. Put a homogenate containing protein X on the column and wash with the starting buffer. Protein X will bind to the column. Then elute by running a salt gradient.

Design an experiment to purify protein X on an anion-exchange column. Protein X has an isoelectric point of 7.

Use a cation-exchange column, such as CM-sepharose, and run it at pH 6. Protein X will have a positive charge and will stick to the column.

Design an experiment to purify protein X on an ion-exchange column. Protein X is only stable between a pH of 6-6.5.

Myelin is a multilayer sheath consisting mainly of lipids (with some proteins) that insulates the axons of nerve cells, facilitating transmission of nerve impulses.

Discuss the structure of myelin and its role in the nervous system.

In the concerted model, all the subunits in an allosteric enzyme are found in the same form, either the T form or the R form. They are in equilibrium, with each enzyme having a characteristic ratio of the T/R. In the sequential model, the subunits change individually from T to R.

Distinguish between the concerted and sequential models for the behavior of allosteric enzymes.

Proteins that are associated with membranes do not have to span the membrane. Some can be partially embedded in it, and some associate with the membrane by non-covalent interactions with its exterior.

Do all proteins associated with membranes span the membrane from one side to another?

Activators make the shape of the curve less sigmoidal.

Does the behavior of allosteric enzymes become more or less cooperative in the presence of activators?

Inhibitors make the shape of the curve more sigmoidal.

Does the behavior of allosteric enzymes become more or less cooperative in the presence of inhibitors?

The presence of a catalysts does affect the rate of the reaction. The standard free energy change is a thermodynamic property that does not depend on the reaction rate. Consequently, the presence of the catalyst has no effect.

Does the presence of a catalyst alter the standard free energy change of a chemical reaction?

greater

Double bonds in fatty acid tails produce kinks in the hydrocarbon chain. Causes disorder in packing against other chains. This disorder causes ____ fluidity in membranes with cis-double bonds versus saturated fatty acid chains.

Cholesterol is not very water-soluble, but lecithin is a good natural detergent, which is actually part of lipoproteins that transport the less soluble fats through the blood.

Egg yolks contain a high amount of cholesterol, but they also contain a high amount of lecithin. From a diet and health amount of lectin. From a diet and health standpoint, how do these two molecules complement each other?

As part of the mechanism, the sodium-potassium ATPase has an aspartate residue that becomes phosphorylated. This phosphorylation alters the conformation of the enzyme and causes it to close on one side of the membrane and open on the other, moving ions in the process.

Explain how phosphorylation is involved in the function of the sodium potassium ATPase.

Sn1 stands for uni-molecular nucleophilic substitution. the uni-molecular part means that it obeys first-order kinetics. If the reaction is R:X + Z: -> R:Z + X:, with an Sn1 reaction, the rate depends on the speed with which the X breaks away from the R. The Z group comes in later nd quickly, compared with the breakdown of R:X. Sn2 stands for bimolecular nucleophilic substitution. This happens with the same reaction scheme if the Z attacks the R:X molecule before it breaks down. Thus, the concentration of both R:X and Z are important, and the rate displays second-order kinetics.

Explain the difference between Sn1 reaction mechanism and an Sn2 reaction mechanism?

unsaturated

Fatty acids that contain one or more C=C double bonds are ________: if contain only C-C single bonds, they are saturated.

even

Fatty acids usually contain _____ numbers of carbons (can contain odd numbers of carbons, depending on how they are biosynthesized)

If the APP is cleaved when already inserted into the cell membrane and not in the endosome, then the attempt to stimulate retromer shuttling of APP from the endosome to the Golgi apparatus will not prove to be a worthwhile approach.

Fighting Alzheimer's disease by boosting the stability of retromers is based on the hypothesis that most of the APP is cleaved in the endosome to release beta amyloid. What if this hypothesis is wrong and most of the APP is cleaved on the cell membrane as previously thought?

K0.5

For an allosteric enzyme, the substrate concentration at one-half Vmax is called the ______.

Use the equation (V = Vmax / (Km + [S])) and the answers are (a) V = 0.5 Vmax (b) V = 0.33 Vmax (c) V = 0.09 Vmax (d) V = 0.67 Vmax (e) V = 0.91 Vmax

For an enzyme that displays Michaelis-Menten kinetics, what is the reaction velocity, V (as a percentage of Vmax)? (a) [S] = Km (B) [S] = 0.5 Km (C) [S] = 0.1 Km (D) [S] = 2 Km (E) [S] = 10 Km

The reaction of glucose with oxygen is thermodynamically favored, as shown by the negative free-energy change. The fact that glucose can be maintained in an oxygen atmosphere is a reflection of the kinetic aspects of the reaction, requiring overcoming an activation-energy barrier.

For the reaction of glucose with oxygen to produce CO2 and H20, Glucose +6O2 --> 6CO2 + 6H2O, the delta G is -2880 kJ mol^-1, a strongly exergonic reaction. However, a sample of glucose can be maintained indefinitely in an oxygen-containing atmosphere. Reconcile these 2 statements

The reaction is first order with respect to A, first order with respect to B, and second order overall. The detailed mechanism of the reaction is likely to involve one molecule each of A and B.

For the reaction, 3A + 2B -> 2C + 3D, the rate was experimentally determined to be rate = k[A]^1[B]^1. What is the order of the reaction with respect to A? With respect to B? What is the overall of the reaction? How many molecules each of A and B are likely to be involved in the detailed mechanism of the reaction?

A protein solution from an ammonium sulfate preparation is passed over a gel-filtration column where the proteins of interest will elute in the void volume. The salt, being very small, will move through the column slowly. In this way, the proteins will leave the salt behind and exit the column without it.

Gel-filtration chromatography is a useful method for removing salts, such as ammonium sulfate, from protein solutions. How is this accomplished?

Lipid-soluble vitamins accumulate in fatty tissue, leading to toxic effects. Water soluble vitamins are excreted, drastically reducing the chances of an overdose.

Give a reason for the toxicity that can be caused by overdoses of lipid-soluble vitamins.

If the protein, once the cells were broken open, you could centrifuge at 100,000 x g, and all the organelles would be in the pellet. Your enzyme would be in the supernatant, along with all the other cytosolic ones.

Give an example of a scenario in which you could partially isolate a protein with differential centrifugation using only one spin?

Most coenzymes are derivatives of compounds we call vitamins. For example, nicotinamide adenine dinucleotide is produced from the B vitamin niacin. Flavin adenine dinucleotide comes from riboflavin.

How are coenzymes related to vitamins?

Enzymes (trypsin and Chymotrypsin) and chemical reagents (Cyanogen bromide CnBr)

How are proteins cleaved?

The families afflicted with a rare form of early onset Alzheimer's disease are prime candidates for studies that attempt to treat the disease before symptoms appear. there are over 500 families participating in clinical studies.

How are researchers working with extended families in Medellin, Columbia to look for methods of fighting Alzheimer's disease?

Cocaine can be degraded by a specific enzyme that hydrolyzes an ester bond that is part of cocaine's structure. In the process of this hydrolysis, the cocaine must pass through a transition state that changes its shape. Catalytic antibodies to the transition state of the hydrolysis of cocaine hydrolyze cocaine to 2 harmless degradation products-benzoic acid and ecgonine methyl ester. When degraded, the cocaine cannot block dopamine reuptake. No prolongation of the neuronal stimulus occurs, and the addictive effects of the drug vanish over time.

How can abzymes be used to treat cocaine addition?

In the case of competitive inhibition, the value of Km increases, while the value of Km remains unchanged in noncompetitive inhibition.

How can competitive and pure noncompetitive inhibition be distinguished in terms of Km?

Lipids can be "tagged" with a fluorescent moiety to observe their motion in membranes. Proteins have intrinsic fluorescence and can be monitored directly.

How can fluorescence techniques be used to monitor the motion of lipids and proteins in membranes?

Within the fractionation range of a gel-filtration column, molecules elute with a linear relationship of log MW versus their elution volumes. A series of standards can be run to standardize the column, and then an unknown can be determined by measuring its elution volume and comparing it to a standard curve.

How can gel-filtration chromatography be used to arrive at an estimate of the molecular weight of a protein?

The graph of rate against substrate concentration is sigmoidal for an allosteric enzyme but hyperbolic for an enzyme that obeys Michaelis-Menten equation.

How can you recognize an enzyme that does not display Michaelis-Menton kinetics?

First homogenize the liver cells using a Potter-Elvehem homogenizer. Then spin the homogenate at 500 X g to sediment the unbroken cells and nuclei. Centrifuge the supernatant at 15,000 x g and collect the pellet, which contains the mitochondria

How could you isolate mitochondria from liver cells using differential centrifugation?

Scientist were taking Acetamidazole to help fight altitude sickness and noticed their beer tasted terrible. They then studied carbonic anhydrase and found it was a chemical sensor for CO2.

How did scientists determine that carbonic anhydrase is a chemical sensor for CO2?

all at the same time

How do changes in the conformation happen in the concerted model?

Whenever there are multiple substrates, the trick is determining the Km of one of them is to run the reaction with saturating concentrations of the other one.

How do scientists determine the Km of a substrate that is part of an ordered reaction with two substrates?

Hexokinase is found predominantly in the muscle and acts during glycolysis of muscle glucose. Glucokinase is found in the liver. The higher the Km of glucokinase can be explained by the body's need to have the muscle enzyme function at lower glucose levels than the liver enzyme under conditions where energy is needed.

How do the Km values for glucokinase and hexokinase reflect their roles in sugar metabolism?

(1) Amino acid analysis, determine which ones are present. (2) Amino acid sequencing, determine the N and C termini and cleave the protein into smaller peptide fragments (most proteins > 100 a.a.)

How do you determine the primary structure of a protein?

Overwhelm the system

How do you get the protein that has bound to the ligand out of the affinity chromatography?

Take the reciprocal of the equation (1/V)= (Km/Vmax[S]) + (1/Vmax)

How do you make a straight line of the Michaelis Menten equation.

Ion exchange chromatography is a specific type of separation based on net charge of the molecules being separated. The term HPLC refers to chromatography procedures carried out under high pressure, but the basis of the separation could be ion exchange, gel filtration, reverse phase, or affinity chromatography.

How does HPLC differ from ion-exchange chromatography?

Valium is an allosteric drug that binds to a different site on the receptors for gama aminobutyric acid (GABA). Valium turns up the response of the receptor for the GABA. When Valium is bound, the response to GABA goes up manifold.

How does Valium work?

Glycogen phosphorylase is controlled allosterically by several molecules. In the muscle, AMP is an allosteric activator. In the liver, glucose is an allosteric inhibitor. Glycogen phosphorylase also exists in a phosphorylated form and an unphosphorylated form, with the phosphorylated form being more active.

How does glycogen phosphorylase is controlled allosterically and by covalent modification.

SDS binds to the protein in a constant ratio of 1.4 g SDS per gram of protein. It coats the protein with negative charges and puts it into a random coil shape. Thus, charge and shape are eliminated.

How does the addition of sodium dodecylsulfate to proteins affect the basis of separation on electrophoresis?

Enzymes are many orders of magnitude more effective as catalysts than are non-enzymatic catalysts.

How does the catalytic effectiveness of enzymes compare with that of non-enzymatic catalysts?

Steroids contain a characteristic fused-ring structure, which other lipids do not.

How does the structure of steroids differ from that of the other lipids discussed in chapter 8?

10^20 times over an uncatalyzed reaction

How fast can enzymes increase the rate of reaction?

Enzymes that exhibit cooperativity do not show hyperbolic curves of rate versus substrate concentration. Their curves are sigmoidal. the level of cooperativity can be seen by the shape of the sigmoidal curve.

How is the cooperative behavior of allosteric enzymes reflected in a plot of reaction rate against substrate concentration?

It is sigmodal, when L (L=T/R) is greater than the curve moves to the right.

How is the curve in the concerted model?

Histidine 57 exists in both the protonated and un-protonated form during the chymotrypsin reaction. Its pKa of 6.0 makes this possible in the physiological pH range.

How is the pKa for histidine 57 is important to its role in the mechanism of chymotrypsin action?

Tunerover number = Vmax/[ET]

How is the turnover number of an enzyme related to Vmax?

Remember the situation with hemoglobin, and think of enzymes similarly. Enzymes that exhibit cooperativity have multiple subunits that can influence each other. Most enzymes that are cooperative exhibit positive cooperatively, which means that the binding of substrate to one subunit will make it easier to bind the substrate to another subunit.

If we describe an enzyme like aspartate transcarbamoylase and say that it exhibits cooperativity, what do we mean?

Pure noncompetitive

If we made a Lineweaver-Burk plot of an irreversible inhibitor, which type of reversible inhibition would it be most likely to resemble?

You may or may not see the same response. As we have seen with aspartase transcarbamoylase, it is possible that one substrate exhibits a hyperbolic response while another one exhibits a sigmoidal response.

If you graph the velocity of an enzyme catalyzed reaction vs [S] for each of the two substrates that are part of a random mechanism would you expect to see the same shape curve? Why or why not?

Those with the highest charge/mass ratio would move the fastest. There are three variables to consider, and most electrophoreses are done in a way to eliminate two of the variables so that the separation is by size or by charge, but not by both.

If you had a mixture of proteins with different sizes, shapes, and charges and you separated them with electrophoresis, which proteins would move fastest toward the anode (positive end)

Reasonably harsh homogenization would be able to liberate the soluble protein X from the peroxisomes, which are fragile. Centrifugation at 15,000 x g would sediment the mitochondria (broken or intact). The supernatant would then have protein X but no protein Y. Freeze/thaw techniques and sonication would accomplish the same thing, or the mitochondria and the peroxisomes could be separated initially by sucrose-gradient centrifugation.

If you had a protein X, which is a soluble enzyme found inside the peroxisome, and you wished to separate it from a similar protein Y, which is an enzyme found embedded in the mitochondrial membrane, what would be your initial techniques for isolating those proteins?

Km, Vmax

In a Lineweaver-Burke plot for Non-competitive inhibition, because the inhibitor does not interfere with binding of the substrate to the active, ____ is unchanged. The _________ does change in the presence of a noncompetitive inhibitor.

slope, y-intercept

In a competitive inhibition, the Lineweaver-Burk double reciprocal plot of 1/V versus 1/[S], the ____ (and the x intercept) changes but the _______ does not change. So, the Km will change, but the Vmax will stay the same in the presence of competitive inhibitor.

Compete

In a competitive inhibition, the substrate must _____ with inhibitor for the active site; more substrate is required to reach a given reaction velocity. EI <--> I + E , K1 = ([E][I]/[EI]). It is an if/or.

The carbon of a carbonyl group is often attacked by a nucleophile.

In biochemistry mechanisms, what group is often attacked by a nucleopile?

The transition temperature is lower in a lipid bilayer with mostly unsaturated fatty acids compared with one with a high percentage of saturated fatty acids. The bilayer with the unsaturated fatty acids is already disordered then the one with a high percentage of saturated fatty acids.

In lipid bilayers, there is an order disorder transition similar to the melting of a crystal. In a lipid bilayer in this transition to occur at a higher temperature, a lower temp, or the same temp as it would in a lipid bilayer in which most of the fatty acids are saturated? Why?

cis isomer

In most unsaturated fatty acids, the _____ ______ predominates; the trans isomer is rare.

The nonpolar amino acids will stick the most to the stationary phase, so glutamic acid will move the fastest, followed by glycine and then phenylalanine.

In reverse-phase HPLC, the stationary phase is nonpolar and the mobile phase is a polar solvent at neutral pH. Which amino acid (phenylalanine, glycine, or glutamic acid) will move the fastest on a reverse-phase HPLC column? Which one is the slowest?

The lecithin in the egg yolks serves as an emulsifying agent by forming closed vesicles. The lipids in the butter (frequently triacylglycerols) are retained in the vesicles and do not form a separate phase.

In the preparation of sauces that involve mixing water and melted butter, egg yolks are added to prevent separation. How do the egg yolks prevent separation? (Hint egg yolks are rich in phosphatidylcholine, which is lecithin)

The surface wax keeps produce fresh by preventing loss of water.

In the produce department of supermarkets, vegetables and fruits (cucumbers are an example) have been coated with wax for shipping and storage. Suggest a reason why this is done?

Uncompetitive

Inhibitor can bind to the ES complex but not to free enzyme (Vmax and Km both decrease)

It is very good, in the case of noncompetitive inhibitors; much of metabolic control depends on feedback inhibition by downstream noncompetitive inhibitors. the question is perhaps moot in the case of competitive inhibitors, which are much less commonly encountered in vivo. Some antibiotics, however, are competitive inhibitors (good for the sick person, bad for the bacteria)

Is it good (or bad) that enzymes can be reversibly inhibited? Why?

Many models are possible. We never really know for sure how the enzyme works, rather, we create a model that explains the observed behavior. It is very possible that another model would do so as well.

Is it possible to envision models for the behavior of allosteric enzymes other than the ones that we have seen in chapter 7?

The term Km should be used for enzymes that display Michaelis-Menten kinetics. Thus, it is not used with allosteric enzymes. Technically, competitive and noncompetitive inhibition are also terms that are restricted to Michaelis-Menten enzymes, although the concepts are applicable to any enzyme. An inhibitor that binds to an allosteric enzyme at the same site as the substrate is similar to a classical competitive inhibitor. One that binds at a different site is similar to a noncompetitive inhibitor, but the equations and the graphs characteristic of competitive and noncompetitive inhibition don't work the same way with an allosteric enzyme.

Is the term Km used with allosteric enzymes? What about competitive and noncompetitive inhibition? Why?

Stereospecificty

It catalyzes a reaction in which one stereoisomer is reacted or formed in preference to all others that might be reacted or formed. Binding sites on enzymes must be asymmetric.

Relative specificity

It catalyzes the reaction of structurally related substances to give structurally related products.

General acid-base catalysis

It depends on the donation and acceptance of protons (proton transfer reactions). Groups that donate or accept protons include imidazole, hydroxyl, carboxyl, sulfhydryl, amino, etc.

Mixed Non-competitive

It is somewhat similar to non-competitive inhibition, but the binding of I does affect the binding of S (Km increases and Vmax decreases).

metal ion

Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes (____ _____ catalysts)

open chains

Lipids include ____ ___ forms and cyclic forms. They include fatty acids, triacylglycerols, sphingolipids, phosphoacylglycerols (phospholipids), and glycolipids. They are also lipid soluble vitamins, prostaglandins, and leukotrienes.

Vitamin A (Serves as the site of the primary photochemical reaction in vision), Vitamin D (regulates calcium and phosphorus metabolism), Vitamin E (serves as an antioxidant; necessary for reproduction in rats and may be necessary for reproduction in humans) Vitamin K (has a regulatory function in blood clotting)

List the fat soluble vitamins, and give a physiological role for each.

Nicotinamide adenine dinucleotide, oxidation-reduction; Flavin adenine dinucleotide, oxidation-reduction; coenzyme A, acyl transfer; pyridoxal phosphate, transamination; biotin, carboxylation; lipoic acid, acyl transfer.

List three coenzymes and their functions?

Prostaglandins and leukotrienes are derived from arachidonic acid. They play a role in inflammation and in allergy and asthma attacks.

List two classes of compounds derived from arachidonic acid. Suggest some reasons for the amount of biomedical research devoted to these compounds.

redox, electron transport chain

NAD+ is used in many ____ reactions in biology. It is very important for the ______ _______ _____ in cellular respiration.

The digestive enzymes trypsin and chymotrypsin are classic examples of regulation by zymogens. The blood clotting protein thrombin is another.

Name the three proteins that are subject to the control mechanism of zymogen activation?

Both the slope and the intercepts will change. The lines will intersect above the x-axis at negative values of 1/[S]

Noncompetitive inhibition is a limiting case in which the effect of binding inhibitor has no effect on the affinity for the substrate and vice versa. What would a Lineweaver-Burk plot look like for an inhibitor that had a reaction scheme similar to a noncompetitive inhibition reaction, but where binding inhibitor lowered the affinity of E1 for the substrate?

Nucleophilic substitution reactions

Nucleophilic (electron-rich) atom attacks electron deficient atom. In biochemistry, the carbon of the carbonyl group (C=O) is often the atom attacked by the nucleophile. Common nucelophiles are the oxygens of serine, threonine, and tyrosine.

Polyacrylamide

Of the two principal polymers used in column chromatography and electrophoresis, which one would be most immune to contamination by bacteria and other organisms?

Primary, secondary

One of the first protein ID techniques designed was the enzyme-linked immunoabsorbent assay (ELISA). ______ antibody binds specifically to protein of interest. _______ antibody allows us to visualize this specific interaction through fluorescence, radioactivity, etc.

The ES complex would be in an "energy trough", with a consequentially large activation energy to the transition state.

Other things being equal, what is a potential disadvantage of an enzyme having a very high affinity for its substrate?

Prostaglandins in blood platelets can inhibit their aggregation. This is one of the important physiological effects of prostaglandins.

Outline a possible connection between the material in chapter 8 and the integrity of blood platelets.

unsaturated

Plant membranes have a higher percentage of ______ fatty acids than animal membranes. Animal membranes are less fluid (more rigid) than plant membranes.

reduces

Presence of cholesterol _______ fluidity by stabilizing extended chain conformations of hydrocarbon tails of the fatty acids.

sodium dodecyl sulfate (SDS)

Prior to running the electrophoresis gel, the protein is treated with detergent ___________ to disrupt the non-covalent interactions for tertiary and quaternary structure.

Solubility properties (insoluble in aqueous or polar solvents, soluble in nonpolar solvents). Some lipids are not at all structurally related.

Proteins, nucleic acids, and carbohydrates are grouped by common structural features found within their group. What is the basis for grouping substances as lipids?

k[A]^1[B]^1

Rate = what?

Both proteins elute in the void volume together and would not be separated.

Sephadex G-75 has an exclusion limit of 80,000 molecular weight for globular proteins. If you tried to use this column material to separate alcohol dehydrogenase (MW 150,000) from beta-amylase (MW 200,000), what would happen?

Yes, the beta-amylase would come out in the void volume, but the bovine serum albumin would be included in the column bead and would elute more slowly.

Sephadex G-75 has an exclusion limit of 80,000 molecular weight for globular proteins. If you tried to use this column material to separate b-amylase (MW 200,000) from bovine serum (MW 66,000), what would happen?

The waxy surface coating is a barrier that prevents loss of water.

Succulent plants from arid regions generally have waxy surface coating. Suggest why such a coating is valuable for the survival of the plant.

The higher percentage of unsaturated fatty acids in membranes in cold climates is an aid to membrane fluidity.

Suggest a reason why animals that live in cold climates tend to have higher proportions of polyunsaturated fatty acid residues in their lipids than do animals that live in warm climates.

Biological membranes are highly nonpolar environments. Charged ions tend to be excluded from such environments rather than dissolving them, as they would have to do to pass through the membrane by simple diffusion.

Suggest a reason why inorganic ions, such as K+, Na+, Ca2+, and Mg2+, do not cross biological membranes by simple diffusion.

Partially hydrogenated vegetable oils have the desired consistency for many foods, such as oleomargarine and components of TV dinners

Suggest a reason why partially hydrogenated vegetable oils are used so extensively in packaged foods.

At a lower temperature, the membrane would tend to be less fluid. the presence of more unsaturated fatty acids would tend to compensate by increasing the fluidity of the membrane compared to one at the same temp with a higher proportion of saturated fatty acids.

Suggest a reason why the cell membranes of bacteria grown at 20 C tend to have a higher proportion of unsaturated fatty acids than the membranes of bacteria of the same species grown at 37 C. In other words, the bacteria grown at 37 C have a higher proportion of saturated fatty acids in their cell membranes.

Nature chooses what works. This is an efficient use of a large protein and of the energy of ATP.

Suggest a reason why the same protein system moves both sodium and potassium ions into and out of the cell.

In a protein that spans a membrane, the nonpolar residues are the exterior ones; they interact with the lipids of the cell membrane. The polar residues are in the interior, lining the channel through which the ions enter and leave the cell.

Suppose that you are studying a protein involved in transporting ions in and out of the cells. Would you expect to find the nonpolar residues in the interior or the exterior? Why? Would you expect to find the polar residues in the interior or the exterior? Why?

Chymotrypsinogen

Synthesized and stored in the pancreas. A single polypeptide chain of 245 amino acids residues cross-linked by 5 disulfide bonds. When secreted into the small intestine, the digestive enzyme trysin cleaves a 15 unit polypeptide from the N-terminal end t give pie-chymotrypsin (the active enzyme form).

transition state

The ___ ___ often has a different shape than either the substrate or the product. The "true nature" of it is a chemical species that is intermediate in structure between the substrate and the product.

Lineweaver-Burke y = mx + b or (1/V) = (Km/Vmax) x (1/[S]) + (1/Vmax)

The _______ _______ plot has the form y = mx + b, and is the formula for a straight line.

induced-fit, cooperativity

The change in conformation is induced by the fit of the substrate to the enzyme, as per the ________ model of substrate binding. Sequential model represents __________.

particle separation

The different spin speeds allow for _____ _____: (1) lower speeds result in a pellet containing unbroken cells and nuclei, (2) higher speeds results in a pellet containing mitochondria, lysosomes, and microbodies., (3) highest speeds results in a pellet containing ribosomes, ER, Golgi , and plasma membrane fragments.

The low Km for the aromatic amino indicates that they will be oxidized preferentially.

The enzyme D-amino acid oxidase has a very high turnover number because it is very toxic. The Km for the enzyme is in the range of 1 to 2 mM for the aromatic amino acids and in the range of 15 to 20 mM for amino acids such as serine, alanine, and the acidic amino acids. Which of these amino acids are the preferred substrates for the enzymes?

The easiest way to follow the rate of reaction is to monitor the decrease in absorbance at 340 nm, reflecting the disappearance of NADH.

The enzyme lactate dehydrogenase catalyzes the reaction, Pyruvate + NADH + H+ -> lactate + NAD+. NADH absorbs light at 340 nm in the near UV region of the electromagnetic spectrum, but NAD+ does not. What experimental method could be used for following this reaction?

First order kinetics

The rate depends on the concentration of the substrate.

Zero order kinetics

The rate does not depend on the concentration of the substrate.

Lower

The ratio of dissociation constants in a concerted model is called c (Kr/Kt). Because _______ c means higher affinity between substrate and R form, so the curve is more sigmoidal.

Isoelectric focusing

The separation is based on differing isoelectric points (pI) of proteins. The charge on the protein changes as it migrates because of the changing pH. When the protein gets to its pI, it has no charge and stops migrating.

phosphate esters, membrane transport

The side chain -OH groups of Ser, Thr, and Tyr can form ______ _____. Phosphorylation by ATP can convert an inactive precursor into an active enzyme. _____ _____ is a common example. (NA+ - K+ pump)

Leukotrienes

They are found in white blood cells, consists of 3 conjugated double bonds, and an important property is constriction of smooth muscles, especially in the lungs.

Phosphoacylglycerols (phospholipids)

They are the 2nd most abundant group of naturally occurring lipids, and they are found in plant and animal cell membranes.

Sphingolipids

They contain sphingosine, which is a long chain amino alcohol. Found in plants and animals. Abundant in cell membranes in the nervous system. Bares structural similarity to phospholipids.

homogenization

Tissue sample containing the protein of interest is subjected to ____, to break open the cells, or lyse them.

The assumption that the Km is an indication of the binding affinity between the substrate and the enzyme is valid when the rate of dissociation of the ES complex to product and enzyme is much smaller than the rate of dissociation of the complex to enzyme substrate.

Under what conditions can we assume that Km indicates the binding affinity between substrate and enzyme?

lower

Unsaturated fatty acids have _____ melting points than their saturated counterparts; the greater the degree of unsaturation, the lower the melting point

Serine, threonine, and tyrosine are the 3 most often phosphorylated amino acids in proteins that are acted upon by kinases. Aspartate is another one that is often phosphorylated.

What amino acids are often phosphorylated by kinases?

Caspases are a family of homodimer cysteine proteases responsible for many processes in cell biology, including apoptosis, signaling within the immune system, and stem cell differentiation.

What are caspases?

Cholesterol, steroid hormones, and bile acids

What are cyclic forms of a lipid?

Isoprene units are 5-carbon moieties that play a role in the structure of a number of natural products, including fat-soluble vitamins.

What are isoprene units? What do they have to with the material in chapter 8?

TAMLs are used to detoxify natural and synthetic pollutants.

What are some of the ways that TAMLs are used?

The allosteric effect can be faster because it is based on simple binding equilibrium. For example, if AMP is an allosteric activator of glycogen phosphorylation effect requires the hormone cascade beginning with glycogen phosphorylase to become more active and to provide energy for the contracting muscles. The phosphorylation effect requires the hormone cascade beginning with glucogen phosphorylase is phosphorylated, so the response time is slower. However, the cascade effect produces many more activated phosphorylase molecules, so the effects are longer and stronger.

What are some possible advantages to the cell in combining phosphorylation with allosteric control?

The protein antibody complexes can be seen based on the nature of the tag carried by the secondary antibody. This tag may be an enzyme that produces a visible color when provided its substrates, a fluorescent marker, or a radioactive compound.

What are some ways that the antibody-protein complex can be visualized in ELISA or western blots?

Metal ions (Lewis acids that behave as coordination compounds Zn2+ or Fe2+) and Coenzymes (organic compounds, many of which are vitamins or are metabolically related to vitamins)

What are the 2 classes of cofactors?

R or relaxed which binds the substrate tightly, and is the active form. The T or taut which binds the substrate less tightly, and is the inactive form.

What are the 2 conformations of the concerted model?

Serine and histidine are the 2 most critical amino acids in the active site of chymotrypsin.

What are the 2 essential amino acids in the active site of chymotrypsin?

The concerted model and the sequential model

What are the 2 principal models for the behavior of allosteric enzymes?

Structural, expression, and interaction

What are the 3 types of proteomics?

The first step in a western blot is the separation of proteins via electrophoresis. The next step takes the gel from the electrophoresis and transfers the proteins from the gel onto a thin membrane of nitrocellulose or other absorbing compound. Once the proteins are transferred, they are incubated with the primary antibody. Lastly, the bands are made visible by reacting with the substrates for the secondary antibody enzyme tag, or they are visualized with a visualized with a fluorometer or X-ray paper.

What are the main procedures involved in a western blot?

Leukotrienes are carboxylic acids with 3 conjugated double bonds.

What are the main structural features of leukotrienes?

Prostaglandins are carboxylic acids that include a 5 membered ring in their structure.

What are the main structural features of prostaglandins?

(1) The protein is run on an electrophoresis gel. (2) It is transferred from the gel onto a thin membrane of nitrocellulose (or other absorbent material). (3) The membrane is incubated with primary antibody. (4) A secondary antibody is used to visualize the protein.

What are the steps in the Western Blot?

Waxes are esters of long chain carboxylic acids and long-chain alcohols. They tend to be found as protective coatings.

What are the structural features of waxes? What are some common uses of compounds of this type?

Ordered, random, and ping-pong

What are the three most common mechanisms for enzyme catalyzed reactions that have two substrates?

Stationary (packed inside a column; samples interact with this phase a gel like matrix) and Mobile (flows over the stationary phase and carries along with it the sample to be separated, it is called the eluent)

What are the two phases of column chromatography?

First, allosteric effectors modulate the response in a more subtle way than orthosteric ones. Second, an allosteric drug is mores specific for one or a few receptor types. Third, allosteric drugs can be safer because they have no effect at all unless the natural ligand is present.

What are three advantages of using allosteric drugs opposed to orthosteric ones?

Trypsin, chymotrypsin, and thrombin are all proteases. trypsin cleaves peptide bonds where there amino acids with positively charged side chains (Lys and Arg). Chymotrypsin cleaves peptides at amino acids with aromatic side chains. Thrombin cleaves the protein fibrinogen into fibrin.

What are three proteases and their substrates?

Electrospray ionization (ESI-MS) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS).

What are two principal types of mass spectrometry?

One is Amgen's Cincalcet, a drug designed to fight chronic kidney failure by improving the action of calcium receptors. The other is an HIV medication by Pfizer called Maraviroc. It interferes with HIV entry into the cells.

What are two recent allosteric drugs that are currently on the market? What do they do?

The basis of most resins is agarose, cellulose, dextran, or polyacrylamide.

What are two types of compounds that make up the resin for column chromatography?

A compound can be eluted by raising the salt concentration or by adding a mobile ligand that has a higher affinity for the bound protein than the stationary resin ligand does. Salt is cheaper but less specific. A specific ligand may be more specific, but is likely to be expensive.

What are two ways that a compound can be eluted from an affinity column? What could be the advantages or disadvantages of each?

A compound can be eluted by raising the salt concentration or by changing the pH. Salt is cheap, but it might not be more specific for a tight pI range, but extremes of pH may also denature the protein.

What are two ways that a compound can be eluted from an ion-exchange column? What could be the advantages or disadvantages of each?

denaturation

What can be the result of increasing the temp too much in a reaction?

With a quaternary amine, the column resin always has a net positive charge, and you don't have to worry about the pH of your buffer altering the form of the column. With a tertiary amine, there is a dissociable hydrogen, and the resin may be positive or neutrally charged, depending on the buffer pH.

What could be an advantage of using an anion exchange column based on a quaternary amine (i.e. resin-N+(CH2CH3)3) as opposed to a tertiary amine (resin-NH+(CH2CH3)2)?

Their amino acid content and arrangements make some proteins more soluble than others. A protein with more highly polar amino acids on the surface is more soluble than one with more hydrophobic ones on the surface.

What differences between proteins are responsible for their differential solubility in ammonium sulfate?

Disruption of apoptosis can lead to forms of cancer and unwanted cell death, such as cells surrounding neurons that have died from a stroke.

What diseases are linked to apoptosis?

Sodium dodecyl-sulfate polyacrylamide gel-electrophoresis. With SDS-PAGE, the charge and shape differences of proteins are eliminated so that the only parameter determining the migration is the size of the protein.

What does SDS-PAGE stand for? What is the benefit of using it?

it is unstable

What does an increase in delta G mean?

The action of a number of receptor proteins requires binding of GTP to a specific subunit, followed by subsequent hydrolysis.

What does the hydrolysis of GTP have to do with the actions of receptor proteins?

activation energy or delta G

What does the rate of reaction depend on?

The presence of a catalyst lowers the activation energy of a reaction.

What effect does a catalyst have on the activation energy of a reaction?

The 2 enzymes associated with the disease are called beta-secretase and gamma-secretase

What enzymes are known to be involved in Alzheimer's disease?

Allosteric enzymes display sigmoidal kinetics when rates are plotted versus substrate concentration. Michaelis-Menten enzymes exhibit kinetics. Allosteric enzymes usually have multiple subunits, and the binding of substrates or effector molecules to one subunit changes the binding behavior of the other subunits.

What features distinguish enzymes that undergo allosteric control from those that obey the Michaelis-Menten equation?

Alzheimer's begins with the buildup of ABeta, which is cut from the APP. In the first step, the enzyme eta-secretase cuts APP outside the cell membrane. then the gamma-secrete enzyme cuts the remaining portion of the APP inside the membrane, releasing ABeta

What happens according to the "amyloid cascade hypothesis?"

Binding of the hormone causes a movement of the transmembrane protein that is transmitted to the intracellular side. This causes a movement of the kinase and catalytic domains, activating the kinase activity of the receptor.

What happens to human growth hormone receptor when it binds hGH?

Scientists looked for drugs that would mimic the behavior of signaling molecules, such as hormones and neurotransmitters.

What has been the historical method used in drug design?

Ion exchange chromatography

What has interactions that are based on overall charge? It has two types; cation exchange (negatively charged) and anion (positively charged).

JAK2 stands for Janus Kinase 2. The hormone binding to the outside of the receptor causes an activation of the kinase on the inside of the cell. Activation of this and many kinases is the key signaling event in many pathways. Such kinases produce a second messenger that begins a cascade of molecular changes that effect overall changes seen when hormones are released.

What is JAK2 and why is it important to the effect of hGH?

A K system is an allosteric enzyme in which the binding of inhibitor alters the apparent substrate concentration needed to reach one-half Vmax, S0.5

What is a K system?

A V system is an allosteric enzyme in which the binding of inhibitor changes the Vmax of the enzyme but not the S0.5

What is a V system?

They are allosteric interactions that occur when several identical molecules are bound to a protein. The binding of substrate molecules to different sites on an enzyme, such as the binding of aspartate to ATCase, is an example of a homotropic effect. Heterotropic effects are allosteric interactions that occur when different substances (such as inhibitor and substrate) are bound to the protein. In the ATCase reaction, inhibition by CTP ad activation by ATP are both heterotropic effects.

What is a homotropic effect? What is a heterotropic effect?

Enzymes tend to have fairly sharp pH optimum valves. It is necessary to ensure that the pH of the reaction mixture stays at the optimum valve. This is especially true for reactions that require or produce hydrogen ions.

What is a reason for carrying out enzymatic reactions in buffer solutions?

A retromer is a molecular complex that shuttles proteins from the endosome to the Golgi apparatus.

What is a retromer?

Coenzymes can accomplish the same mechanisms that the amino acids do in a reaction. For example, a metal ion may act as a general acid or base. Parts of a coenzyme, such as the reactive carbanion of thiamine pyrophosphate, may act as a nucleophile to catalyze the reaction.

What is a role for coenzymes based on reaction mechanisms?

It is a substrate that binds irreversibility to the active site, permanently inactivating the enzyme. They are important because they are used as potent drugs to knock out an enzyme, and they are used to study enzyme kinetics with a focus on interactions at the active site.

What is a suicide substrate? Why are they important?

The advantages of an ELISA would be ease of use, low cost, and ready availability to any researcher. The disadvantages are that, compared to a microarray, relatively few proteins can be tested at one time. A microarray can test thousands of proteins in a single experiment, so it is much more powerful. However, it is much more expensive and requires specialized equipment not readily available.

What is an advantage of using an ELISA instead of a protein microarray to study a proteome? What is a disadvantage?

Apoptosis is a natural phenomenon of programmed cell death.

What is apoptosis?

Green chemistry refers to the modern techniques that replace large quantities of toxic chemicals previously used smaller quantities of less toxic chemicals.

What is green chemistry?

General acid catalysis is the part of an enzyme mechanism in which an amino acid or other molecule donates a hydrogen ion to another molecule.

What is meant by general acid catalysis with respect to enzyme mechanisms?

Salting out is a process whereby a highly ionic salt is used to reduce the solubility of a protein until it comes out of solution and can be centrifuged. The salt forms ion-dipole bonds with the water in the solution, which leaves less water available to hydrate the protein. Nonpolar side chains begin to interact between protein molecules, and they become insoluble.

What is meant by salting out? And how does it work?

Proteomics is the systematic analysis of an organism's complete complement of proteins, or its proteome. Just as we learned the basic dogma of molecular biology (DNA -> RNA -> protein), the technology now available has allowed scientists to describe has allowed scientists to describe all the DNA of an organism as its genome, all of the RNA as its transcriptome, and all of the proteins produced as its proteome. To understand the flux of proteins in a cell is to understand its metabolism.

What is proteomics?

Km = 7.42 mM, Vmax = 15.9 mmol min^-1, noncompetitive inhibition

What is the Km and is the inhibition competitive or noncompetitive? [S] = 0.0001, 0.00005, 0.0015, 0.0025, 0.005 V with no inhibitor = 0.026, 0.092, 0.136, 0.15, 0.165 V with inhibitor = 0.01, 0.04, 0.086, 0.12, 0.142

Proteins are transferred to nitrocellulose because all of the proteins ends up layered on top of a very thin membrane. This means that small volumes of the antibody solutions can be used to bind proteins. These antibodies are very expensive, so the less used, the better. Also, since the original proteins were imbedded in a gel, if the gel were reacted directly with the antibodies, the antibodies would not have easy access to the gel, as they would not percolate easily through the pores of the gel, as they would not percolate easily through the pores of the gel without the benefit of an electric current to push them.

What is the advantage to transferring proteins bands from a gel to nitrocellulose during the western blot?

(A) size (B) Specific ligand-binding ability (C) Net charge (D) Polarity

What is the basis for the separation of proteins by the following techniques? (A) gel-filtration chromatography. (B) affinity chromatography. (C) ion-exchange chromatography. (D) reverse phase HPLC

ELISA is based on antibody-protein interactions. Specific antibodies, called primary antibodies, are put into microtiter plates to localize target proteins. A secondary antibody carrying some type of tag to render it visible is also added. If the target protein is there, then the combination of the protein-primary antibody-secondary antibody will be visible.

What is the basis for the technique called ELISA?

With a ping-pong mechanism, one product is released prior to the binding of the second substrate. With the other 2, both substrates are bound before any product is released.

What is the biggest difference between a ping-pong mechanism and either an ordered mechanism or random mechanism?

A primary antibody is specific for a target protein that a researcher is looking for. A secondary antibody will react with the primary antibody. The secondary antibody carries the tag that makes the complex visible.

What is the difference between a primary and a secondary antibody?

In the lock and key model, the substrate fits into a comparatively rigid protein that has an active site with a well defined shape. In the induced fit model, the enzyme undergoes a conformational change on binding to the substrate. The active site takes shape around the substrate.

What is the difference between lock-and-key and induced-fit models for binding of a substrate to an enzyme?

With pure noncompetitive inhibition, the binding of the inhibitor des not change the affinity of the enzyme for substrate at all and vice versa; thus the Km does not change. With mixed inhibition, the substrate and inhibitor doe affect each other such that the Km for the substrate is different in the presence of inhibitor.

What is the difference between pure and mixed noncompetitive inhibition?

A competitive inhibitor binds to the active site of an enzyme, preventing binding of the substrate. A noncompetitive inhibitor binds at ta site different from the active site, causing a conformational change, which renders the active site less able to bind substrate and convert it to product.

What is the difference between the molecular mechanisms of competitive and noncompetitive inhibition?

Hydrophobic interactions among the hydrocarbon tails are the main energetic driving force in the formation of lipid bilayers.

What is the energetic driving force for the formation of phospholipid bilayers?

A mercury compound was used to separate the subunits of ATCase. When the subunits were separated, one type of subunit retained catalytic activity but was no longer allosteric and was not inhibited by CTP. The other subunit type had no ATCase activity, but it did bind to CTP and ATP.

What is the experiment used to determine the structure of ATCase? What happens to the activity and regulatory activities when the subunits are separated?

A kinase is an enzyme that phosphorylates a protein using a high-energy phosphate, such as ATP, as the phosphate donor.

What is the function of a protein kinase?

Histidine 57 performs a series of steps involving general base catalysis followed by general acid catalysis. In the first phase, it takes a hydrogen from serine 195, acting as a general base. This is followed immediately by an acid catalysis step, in which it gives the hydrogen to the amide group of the peptide bond that is breaking. A similar scheme takes place in the second phase of the reaction.

What is the function of histidine 57 in the mechanism of chymotrypsin?

In most chromatography systems, the ligands and solvents are polar,. In reverse phase HPLC, a solution of nonpolar compounds is put through a column that has a nonpolar liquid immobilized on an inert matrix. A more polar liquid serves as the mobile phase and is passed over the matrix. The solute molecules are eluted in proportion to their solubility in the more polar liquid.

What is the main difference between reverse phase HPLC and standard ion-exchange or gel filtration chromatography?

Side effects occur because the drug that is meant to affect one type of receptor will likely have unintended effects on several others.

What is the main reason for side effects with traditional drugs that bind to the active site of a receptor?

hydrophobic interaction

What is the major force driving the formation of lipid bilayers?

It is an enzyme used in the early stages of cytidine nucleotide synthesis.

What is the metabolic role of asparate transcarbamoylse?

Prion disease are transmissible, while the other neurodegenerative disease like Alzheimer's are not.

What is the most significant difference between prion diseases and other diseases caused by amyloid-type plaques, such as Alzheimer's disease?

Salicylic acid, which comes from the bark of the willow tree.

What is the natural compound that eventually led to aspirin?

The largest proteins elute first; the smallest elute last. Larger proteins excluded from the interior of the gel bead so they have less available column space to travel. Essentially, they travel a shorter distance and elute first.

What is the order of elution of proteins on a gel-filtration column? Why is this so?

There are thousands of primary antibodies that can be purchased commercially. Other research requires a new primary antibody to be created. The process of attaching an enzyme, a fluorescent marker, or a radioactive compound is a long and difficult task. If every primary antibody had to be tagged, it would be a daunting exercise for the company trying to do it. Instead, a company can specialize in tagging a secondary antibody directed against goat, rabbit, or mouse antibodies. These secondary antibodies can then be used for any experiment where the primary antibody came from one of these animals. Also, there is a signal multiplication effect of using a primary antibody since several secondary antibodies will attach to a single molecule of primary antibody.

What is the point to labeling a secondary antibody with a marker that can be visualized instead of just labeling the primary antibody?

Retromers are thought to play a role in the processing APP. If they are working too slowly, then not enough of the APP makes it into the cell membranes and the cell has more time for the enzymes to cleave the protein into Alpha-beta

What is the proposed role of retromers in Alzheimer's disease?

The induced-fit model assumes that the enzyme and substrate must both move and change to conform to each other perfectly. Thus, the true fit is not between the enzyme and substrate but between the enzyme and the transition state of the substrate on its way to product. A transition-state analogue fits the enzyme nicely in this model.

What is the relationship between a transition-state analog and the induced fit model of enzyme kinetics?

Salicylate stimulates AMPK, which stimulates fat burning. Researchers believe this effect lowers plasma fatty acids and reduces the risk of heart attacks and type 2 diabetes.

What is the relationship between the action of salicylate or aspirin that researchers believe accounts for some of its therapeutic effects?

Any combination of fatty acids is possible.

What is the structural formula for a triacylglycerol, and what is the name of the component parts?

Cholesterol is a precursor of vitamin D3; the conversion reaction involves ring opening.

What is the structural relationship between vitamin D3 and cholesterol?

ATCase is made up of two different types of subunits. One of them is the catalytic subunit, and there are six of them organized into 2 trimers. The other is the regulatory subunit, which consists of 6 protein subunits organized into 3 dimers.

What is the structure of ATCase?

ATP acts as a positive effector of ATCase, and CTP acts as an inhibitor.

What molecule acts as a positive effector (activactor) of ATCase? What molecule acts as an inhibitor?

Replication of the HIV genome inside an infected cell via inhibition of the HIV integrase.

What part of the HIV lifecycle is disrupted by MK-0518?

The production of new virus particles inside the infected cell via inhibition of the HIV protease.

What part of the HIV lifecycle is disrupted by the drugs indinavir and amprenavir?

Size, shape, and charge

What physical parameters of a protein control its migration on electrophoresis?

They act as Lewis acids (electron-pair acceptors) and can take part in enzyme catalysis mechanisms of enzymes.

What properties of metal ions make them useful cofactors?

Amyloid beta and theta are the 2 proteins that form destructive plaques. The former is formed from pieces cut from a precursor protein called amyloid precursor protein.

What proteins are involved in the formation of the destructive plaques found in Alzheimer's disease?

Phosphorylation of tyrosine residues can activate or deactivate a receptor protein, depending on the specific system.

What role does phosphorylation of tyrosine residues play in the action of receptor proteins?

Size-exclusion (gel-filtration) chromatography

What separates molecules based on size? The stationary phase is composed of cross-linked gel particles and packed into a column, and the extent of this can be controlled to determine pore size. Smaller molecules are caught in the pores and larger ones skip the pores and are eluted first.

Both sphingomyelins and phosphatidylcholines contain phosphoric acid esterified to an amino alcohol, which must be choline in the case of a phosphatidylcholine and may be choline in the case of a sphingomyelin. They differ in the 2nd alcohol to which phosphoric acid is esterified. In phosphatidylcholines, the 2nd alcohol is glycerol, which has also formed ester bonds to 2 carboxylic acids. In sphingomyelins, the 2nd alcohol is an another amino alcohol, sphingosine, which has formed an amide bond to a fatty acid.

What structural features do a sphingomyelin and a phosphatidyl choline have in common? How do the structures of these two types of lipids differ?

In both types of lipids, glycerol is esterified to carboxylic acids, with 3 such ester linkages formed in triacylglycerols and 2 in phosphatidyl ethanolamines. The structural difference comes in the nature of the 3rd ester linkage to glycerol. In phosphatidyl ethanolamines, the 3rd hydroxyl group of glycerol is esterified not to a carboxylic acid but to phosphoric acid. The phosphoric acid moiety is esterified in turn to ethanolamine.

What structural features do a triacylglycerol and a phosphatidyl ethanolamine have in common? How do the structures of these two types of lipids differ?

Sphingolipids contain amide bonds, as do proteins. Both can have hydrophobic and hydrophilic parts, and both can occur in cell membranes, but their functions are different.

What structural features does a sphingolipid have in common with proteins? Are there functional similarities?

DNA is the molecule most often separated on agarose electrophoresis, although proteins can also be separated.

What type of macromolecules are usually separated on agarose electrophoresis gels?

Vitamin B6 is the source of pyridoxal phosphate, which is used in transamination reactions.

What type of reaction uses vitamin B6?

Agarose and polyacrylamide

What types of compounds make up the gels used in electrophoresis?

Using a blender, a Potter-Elvejhem homogenizer, or a sonicator

What types of homogenization techniques are available for solubilizing a protein?

It might tell you if the protein were pure or if there were subunits.

What useful information might you get if you did determine the N-terminal amino acid as a separate step?

Affinity chromatography

What uses specific binding properties of molecules or proteins that specifically bind to ligands?

size

What usually determines the mobility of protein on an electrophoresis? Charge, size, or shape?

A good transition state analogue would have to have a tetrahedral carbon atom where the amide carbonyl group was originally found, since the transition state involves a momentary tetrahedral form. It would also have to have oxygens on the same carbon, so that there would be sufficient specificity for the active site.

What would be the characteristics of a transition-state analog for the chymotrypsin reaction?

In the first cycle, the first and second amino acids from the N-terminal end would be reacted and released as PTH derivatives. You would get a double signal and not know which one was the true N-terminus.

What would happen during an amino acid sequencing experiment using the Edman degradation if you accidently added twice as much Edman reagent (on a per-mole basis) as the peptide you were sequencing?

Under conditions of low substrate concentration.

When does the kcat/Km value approximate the catalytic efficiency of an enzyme?

The removal of the oil also removes the natural oils and waxes on the feathers. These oils and waxes must regenerate before the birds can be released.

When water birds have had their feathers fouled with crude oil after an oil spill, they are cleaned by rescuers to remove the spilled oil. Why are they not released immediately after they are cleaned?

The Lineweaver-Burk line for the enzyme plus inhibitor would angle in the other direction from the uninhibited compared to normal.

When we compare the binding of I and of S to the enzyme in a mixed noncompetitive inhibitor, we assumed that the binding of I decreased the affinity of the enzyme for S. What would happen if the opposite was true?

If you need to maintain the structural integrity of the subcellular organelles, a Potter-Elvejhem homogenizer would be better because it is more gentle. The tissue, such as liver, must be soft enough to use with this device.

When would you choose to use a Potter-Elvehejem homogenizer instead of a blender?

Western blot got its name as a humorous derivation from the original blotting technique called Southern blotting. The original blotting technique was for DNA and was developed by a researcher named Southern, so they called the technique the Southern blot. The next type of molecule to be blotted was RNA, so to distinguish from a Southern blot, it was called a northern blot. After that, the technique for blotting protein was developed and called a western blot.

Where did western blot get its name?

In a Lineweaver-Burk plot for competitive inhibition, the lines intersect at the y-axis intercept, which is equal to 1/Vmax. In a Lineweaver-Burk plot for noncompetitive inhibition, the lines intersect at the x-axis intercept, which equals to -1/Km.

Where do lines intersect on a Lineweaver-Burk plot showing competitive inhibition? On a Lineweaver-Burk plot showing noncompetitive inhibition?

The sequential model can explain negative cooperativity, because a substrate binding to the T form could induce other subunits to switch to the T form, thereby reducing binding affinity.

Which allosteric model can explain negative cooperativity?

Phospholipids are more hydrophilic than cholesterol. the phosphate group is charged, and the attached alcohol is charged and polar. These groups interact readily with water. Cholesterol has only a single polar group, an -OH.

Which is more hydrophilic, cholesterol or phospholipids? Why?

(C) Triacylglycerol is not found in animal membranes.

Which of the following lipids are not found in animal membranes? (A) Phosphoglycerides (B) Cholesterol (C) Triacylglycerols (D) Glycolipids (E) Sphingolipids

(C) and (D) are consistent with what is known about membranes. Covalent bonding between lipids and proteins {statement (E)} occurs in some anchoring motifs, but is not widespread otherwise. Proteins "float" in the lipid bilayers rather than being sandwiched between them {statement (A)}. Bulkier molecules tend to be found on the outer lipid layer {statement (B)}.

Which of the following statements is (are) consistent with what is known about membranes? (A) A membrane consists of a layer of proteins sandwiched between two layers of lipids. (B) The compositions of the inner and outer lipid layers are the same in any individual membrane. (C) Membranes contain glycolipids and glycoproteins. (D) Lipid bilayers are an important component of membranes. (E) Covalent bonding takes place between lipids and proteins in most membranes.

Statements (C) and (D) are correct. Traverse diffusion is only rarely observed {statement (B)}, and term mosaic refers to the pattern of distribution of proteins in the lipid bilayer {statement (E)}. Peripheral proteins are also considered part of membrane {statement (A)}.

Which of the statements are consistent with the fluid mosaic model of membranes? (A) All membrane proteins are bound to the interior of the membrane. (B) Both proteins and lipids undergo transverse (flip-flop) diffusion from the inside to the outside of the membrane. (C) Some proteins and lipids undergo lateral diffusion along the inner or outer surface of the membrane. (D) Carbohydrates are covalently bonded to the outside of the membrane. (E) The term mosaic refers to the arrangement of the lipids alone.

The Sn1 reaction leads to loss of stereospecificity as the X group leaves before the entering nucleophile. This means that the nucleophile can enter from different angles, leading to different isomers.

Which of the two reaction mechanisms in the biochemistry mechanism (which are attacked by a nucleopile) is likely to cause the loss of stereospecificity? Why?

Statements (A) and (C) are correct; statement (B) is not correct because ions are larger molecules, especially polar ones, require channel proteins.

Which statement are consistent with the known facts about membrane transport? (A) Active transport moves a substance from a region in which its concentration is lower to one in which its concentration is higher. (B) Transport does not involve any pores or channels in membranes. (C) Transport proteins may be involved in bring substances into cells.

Vitamin A and E are known to scavenge free radicals, which can do oxidative damage to cells.

Why are many vitamin supplements sold as antioxidants? How does this relate to the material in chapter 8?

Cocaine blocks the reuptake of the neurotransmitter dopamine at synapses. Thus, dopamine stays in the system longer, over-stimulating the neuron and leading to the reward signals in the brain that lead to addiction. Using a drug to block a receptor would be of no use with cocaine addiction and would probably just make removal of dopamine even more unlikely.

Why can cocaine addition not be treated with a drug that blocks the cocaine receptor?

Vitamin K plays a role in the blood clotting process. Blocking its mode of action can have an anticoagulant effect.

Why can some vitamin-K antagonists act as anticoagulants?

The amount of Edman reagent must exactly match the amount of N-termini in the first reaction. If there is too little Edman reagent, some of the N-termini will not react. If there is too much, some of the second amino acid will react. In either case, there will be a small amount of contaminating phenylthiohydantoin (PTH) derivatives. This error grows with the number of cycles run until the point that two amino acids are released in equal amounts, and you cannot tell which one was supposed to be the correct one.

Why can the Edman degradation not be used effectively with very long peptides? (Think about the stoichiometry of the peptides and the Edman reagent and the percent yield of the organic reactions involving them)

Because the inhibitor can bind to E or to ES equally well, any time there is inhibitor present, some of the enzyme will be tied up in the EIS form, which does not lead to catalysis. For this reason it would appear that less enzyme is present.

Why can we say that having a pure noncompetitive inhibitor present is similar to just having less enzyme present?

Raising the salt concentration is relatively safe. Most proteins will elute this way, and , if the protein is an enzyme, it will still be active. If necessary, the salt can be removed later via dialysis. Changing the pH enough to remove the charge can cause the proteins to become denatured. Many proteins are not soluble at the isoelectric points.

Why do most people elute bound proteins from an ion-exchange column by raising the salt concentration instead of changing the pH?

A competitive inhibitor blocks binding, not catalysis.

Why does a competitive inhibitor not change Vmax?

A noncompetitive inhibitor does not change the affinity of the enzyme for its substrate.

Why does a pure noncompetitive inhibitor not change the observed Km?

Acetamidazole is an inhibitor of carbonic anhydrase, which is part of taste receptor that responds to CO2.

Why does acetazolamide make beer taste flat?

Chymotrypsinogen is an inactive zymogen. It is acted upon by trypsin, which cleaves peptides at basic residues, like arginine. When trypsin cleaves between the arginine and the isoleucine, chymotrypsinogen becomes semi-active, forming pie chymotrypsin. This molecule digests itself further, forming the active alpha chymotrypsin. As it turns out, the alpha amino group of the isoleucine produced by the first cleavage is near the active site of alpha chymotrypsin and necessary for its activity.

Why does cleavage of the bond between arginine 15 and isoleucine 16 of chymotrypsinogen activates the zymogen.

Less heart disease is associated with diets low in saturated fatty acids.

Why does the American Heart Association recommend the use of canola oil or olive oil rather than coconut oil in cooking?

The binding of inhibitor to the ES complex to form EIS removes some of the ES. By LeChatelier's principle, this will tend to force the reaction to the right forming more ES. By stimulating the binding of E and S in this manner, the graph will show that the Km is reduced.

Why does the apparent Km decrease in the presence of an uncompetitive inhibitor?

The initial phase release the first product and involves an acylenzyme intermediate. This step is faster than the second part, in which water comes into the active site and breaks the acyl-enzyme bond.

Why does the enzyme reaction for chymotrypsin proceed in 2 phases?

Zymogens are often seen with digestive enzymes that are produced in one tissue and used in another. If the enzyme were active immediately upon production, it would digest other cell proteins, where it would cause great damage. By having it produced as a zymogen, it can be safely made and then transported to the digestive tissue, such as the stomach or small intestine, where it can then be activated.

Why is it necessary or advantage for the body to make zymogens?

This allows for a more rapid response when the hormone is needed. the hormone is already synthesized and usually just requires breaking one or two bonds to make it active. the hormone can be poised and ready to go on demand.

Why is it necessary or advantageous for the body to make inactive hormone precursors?

The Edman degradation will give the identity of the N-terminal amino acid in its first cycle, so doing a separate experiment is not necessary.

Why is it no longer considered necessary to determine the N-terminal amino acid of a protein as a separate step?

Vitamin D can be made in the body.

Why is it possible to argue that vitamin D is not a vitamin?

It is easier to detect deviations of individual points from a straight line than from a curve.

Why is it useful to plot rate data for enzymatic reactions as a straight line rather than as a curve?

Taking too much Valium is not as deadly as taking too much Phenobarbital because the Valium does not have a direct effect. Rather, it modulates the effect of the bound, natural ligand.

Why is taking too much Valium not as dangerous as taking too much Phenobarbital?

A Lineweaver-Burk plot is useful because it gives a straight line. It is easier to determine how well points fit to a straight line than to a curve.

Why is the Lineweaver-Burk plot useful in analyzing kinetic data from enzymatic reactions?

In a polyacrylamide gel used for gel-filtration chromatography, the larger proteins can travel around the beads, thereby having a shorter path to travel and therefore eluting faster. With electrophoresis, the proteins are forced to go through the matrix, so the larger ones travel more slowly because there is more friction.

Why is the order of separation based on size opposite for gel filtration and gel electrophoresis, even though they often use the same compound to form the matrix?

The first phase is faster for several reasons. the serine at position 195 is a strong nucleophile for the initial nucleophilic attack. It then forms an acyl-enzyme intermediate. In the second phase, water is the nucleophile, and it takes time for water to diffuse to the right spot to perform its nucleophilic attack. It is also not as strong a nucleophile as the serine. Therefore, it takes longer for water to perform its nucleophilic attack and break the acyl-enzyme intermediate than it takes for serine to create it.

Why is the second phase of chymotrypsin mechanism is slower than the first phase?

The public is attuned to the idea of polyunsaturated fats as healthful. The trans configuration gives a more palatable consistency. Recently, however, concerns have arisen about the extent to which such products mimic saturated fats.

Why might some food companies find it economically advantageous to advertise their product (for example, triacylglycerols) as being composed of polyunsaturated fatty acids with trans-double bonds?

Heating a protein denatures it. Enzymatic activity depends on the correct 3D structure of the protein. The presence of bound substrate can make the protein harder to denature.

Why would a heating solution containing an enzyme markedly decrease its activity. Why is the decrease of activity frequently much less when the solution contains high concentration of the substrate?

Beta-secretase has the natural function of being involved in the myelination of nerves.

Why would doctors not want to just completely inhibit beta secretase in a patient with Alzheimer's disease?

The L value is the equilibrium ratio of the T/R form. The c value is the ratio of the dissociation constants for substrate and the 2 forms of enzyme, such that c = Kr/Kt

With respect to the concerted model, what is the L value? What is the c value?

Greater cooperativity is favored by having a higher ratio of the T/R form. It is also favored by having a higher dissociation constant for the substrate binding to the T form.

With the concerted model, what conditions favor greater cooperativity?

To the first question, most probably; local concentration (mass-action concepts) could easily dictate the direction. To the second question, probably not; local concentrations would seldom be sufficient to overcome a relatively large delta G of -5.3 kcal in the reverse reaction.

Would nature rely on the same enzyme to catalyze a reaction either way (forward or backward) if the delta G were -0.8 kcal mol^-1? If it were -5.3 kcal mol^-1?

An irreversible inhibitor is bound by covalent bonds. Non-covalent interactions are relatively weak and easily broken.

Would you expect an irreversible inhibitor of an enzyme to be bound by covalent or non-covalent interactions? Why?

A noncompetitive inhibitor does not bind to the active site of an enzyme. Its structure need bear no relation to that of the substrate.

Would you expect the structure of a noncompetitive inhibitor of a given enzyme to be similar to that of its substrate?

The use of a pH meter would not be a good way to monitor the rate of reaction. You are probably running this reaction in a buffer solution to keep the pH relatively constant. If you are not running the reaction in a buffer solution, you run the risk of acid denaturation of the enzyme.

Would you use a pH meter to monitor the progress of this reaction, Pyruvate + NADH + H+ -> lactate + NAD+. Why or why not?

It is possible that your protein is not pure and needs additional purification steps to arrive at a single polypeptide. It is also possible that the protein has subunits, so multiple polypeptide chains could be yielding the contradictory results.

You are in the process of determining the amino acid sequence of a protein and must reconcile contradictory results. In one trail, you determine a sequence with glycine as the N-terminal amino acid and asparagine as the C-terminal amino acid. In another trial, your results indicate phenylalanine as the N-terminal amino acid and alanine as the C-terminal amino acid. How do you reconcile this apparent contradiction?

Tables exist to tell you how many grams of ammonium sulfate [(NH4)2SO4] to add to get a certain percent saturation. A good plan would be to take the homogenate and add enough ammonium sulfate to yield a 20% saturated solution. Let the sample sit for 15 minutes on ice and then centrifuge. Separate the supernatant to arrive at a 40% saturated solution and repeat the process. In this way, you will find out what the percent saturation in ammonium sulfate needs to be to precipitate the protein.

You are purifying a protein for the first time. You have solubilized it with homogenization in a blender followed by differential centrifugation. You wish to try ammonium sulfate precipitation as the next step. Knowing noting beforehand about the amount of ammonium sulfate to add, design an experiment to find the proper concentration (% saturation) of ammonium sulfate to use.

The number of moles of enzyme is 1.56 x 10^-10. The turnover number is 10,700 sec^-1

You do an enzyme kinetic experiment and calculate a Vmax of 100 micro moles of product per minute. If each assay used 01 mL of an enzyme solution that had a concentration of 0.2 mg/mL, what would be the turnover number if the enzyme had a molecular weight of 128,000 g/mol?

Not all AIDS drugs are enzyme inhibitors, but an important class of such drugs inhibits the HIV protease.

You have been hired by a pharmaceutical company to work on development of drugs to treat AIDS. What information would be useful to you?

This lipid is a ceramide, which is one kind of sphingolipid.

You have just isolated a pure lipid that contains only sphingosine and a fatty acid. To what class of lipids does it belong?

The easiest way would be to use a sucrose gradient to separate the mitochondria from the peroxisomes first. Then break open the mitochondria via harsh homogenization or sonication, and then centrifuge the mitochondria. The pellet would contain protein B, while the supernatant would contain protein A. Contaminants could still exist, but they could be cleaned away by running gel filtration, on sephadex G-75 (which would separate enzyme C from enzymes A and B), and then running ion-exchange chromatography on Q-sepharose at pH 7.5. Enzyme B would be neutral and would elute, while enzyme A would stick to the column

You wish to separate and purify enzyme A from contaminating enzymes B and C. Enzyme A is found in the matrix of the mitochondria. Enzyme B is embedded in the mitochondrial membrane, and enzyme C is found in the peroxisome. A and B have MW of 100,000 Da. A has a pI of 6.5. B and C have pI values of 7.5. How would you separate A from the other two?

Induced fit

___ ___ model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit.

Km

____ is a measure of how tightly the substrate is bound to the enzyme. the greater the value of Km, the less tightly S is bound to E. In most cases, Km can be considered the dissociation constant for the ES complex.

Ammonium sulfate (NH4SO4)

____ is commonly used after homogenization as a first step in protein purification; this is called salting out because it takes away H2O from protein when the salt ions interact with the water.

CnBr

_____ cleaves at the C-terminus of internal methionines.

Vmax

_____ is related to the turnover number (how productive the enzyme is) of an enzyme. This quantity is the constant kcat. Number of moles of substrate that react to form product per mole of enzyme per unit of time. (Vmax/[ET]) = turnover number = kcat.

Trypsin

______ cleaves at the C-terminus of amino acids with positively (+) charged side chains.

Km

______ is the substrate concentration at 1/2 Vmax.

order

______ of reaction: the sum of the exponents in the rate equation. (rate=k[A]^1[B]^1)

Chymotrypsin

________ cleaves at the C-terminus of amino acids with aromatic side chains (contain ring structures)

glycerol

a 3 carbon compound that contains three hydroxyl groups, one bound to each carbon.

SN2

a bimolecular nucleophilic substitution reaction; an important type of organic reaction seen in biochemistry; the rate of reaction follows second-order kinetics.

protein kinases

a class of enzymes that modify a protein by attaching a phosphate group to it.

serine proteases

a class of proteolytic enzymes in which a serine hydroxyl plays an essential role in catalysis.

aspartate transcarbamoylase (ATCase)

a classic example of an allosteric enzyme that catalyzes an early reaction in pyrimidine biosynthesis.

dextran

a complex polysaccharide that is often used in column chromatography resins.

agarose

a complex polysaccharide used to make up resins for use in electrophoresis and in column chromatography.

phosphatidic acid

a compound in which 2 fatty acids and phosphoric acid are esterified to the 3 hydroxyl groups of glycerol.

negative cooperativity

a cooperative effect whereby binding of the first ligand to an enzyme or protein causes the affinity for the next ligand to be lower.

competitive inhibition

a decrease in enzymatic activity caused by binding of a substrate analogue to the active site.

concerted model

a description of allosteric activity in which the conformations of all subunits change simultaneously.

induced fit model

a description of substrate binding to an enzyme such that the conformation of the enzyme changes to accommodate the shape of the substrate.

sequential model

a description of the action of allosteric proteins in which a conformational change in one subunit is passed along to other subunits.

lock and key model

a description of the binding of substrate to an enzyme such that the active site and the substrate exactly match each other in shape.

caspases

a family of homodimer cysteine proteases responsible for many processes.

arachidonic acid

a fatty acid that contains 20 carbon atoms and 4 double bonds, the precursor of prostaglandins and leukotrienes.

column chromatography

a form of chromatography in which the stationary phase is packed in a column.

polyacrylamide

a form of electrophoresis in which a polyacrylamide gel serves as both a sieve and a supporting medium.

noncompetitive inhibition

a form of enzyme inactivation in which a substance binds to a place other than the active site but distorts the active site so that the reaction is inhibited.

reverse phase HPLC

a form of high-performance liquid chromatography in which the stationary phase is non-polar and the mobile phase is a polar liquid.

electrospray ionization (ESI-MS)

a form of mass spectrometry in which the sample is converted to fine droplets by spraying.

cerebroside

a glycolipid that contains sphingosine and a fatty acid in addition to the sugar moiety.

Lineweaver-Burk double-reciprocal plot

a graphical method for analyzing the kinetics of enzyme-catalyzed reactions.

triacylglycerol

a lipid formed by esterification of 3 fatty acids to glycerol; also called a triglyceride.

vitamin E

a lipid soluble antioxidant

glycolipid

a lipid to which a sugar moiety is bonded.

percent recovery

a measurement of the amount of an enzyme recovered at each step of purification experiment

Edman degradation

a method for determining the amino acid sequence of peptides and proteins.

electrophoresis

a method for separating molecules on the basis of the ratio of charge to size.

ion-exchange chromatography

a method for separating substances on the basis of charge.

isoelectric focusing

a method for separating substances on the basis of their isoelectric points.

rhodopsin

a molecule crucial to vision; it is formed by the reaction of retina and opsin.

Michaelis constant

a numerical value for the strength of binding of substrate to an enzyme; an important parameter in enzyme kinetics.

phosphoaclyglycerol

a phosphatidic acid with another alcohol esterified to the phosphoric acid moiety.

affinity chromatography

a powerful column separation procedure based on specific binding of molecules to a ligand.

facilitated diffusion

a process by which substances enter a cell by binding to a carrier protein; this process does not require energy.

differential centrifugation

a process in which ruptured cells are centrifuged several times increasing the force of gravity each time.

rate constant

a proportionality constant in the equation that describes the rate of a reaction

trypsin

a proteolytic enzyme specific for basic amino acid residues as the site of hydrolysis.

chymotrypsin

a proteolytic enzyme that preferentially hydrolyzes amide bonds adjacent to aromatic amino acid residues

chymotrypsin

a proteolytic enzyme that preferentially hydrolyzes amide bonds adjacent to aromatic amino acid residues.

salting out

a purification technique for proteins based on differential solubility in salt solutions.

substrate

a reactant in an enzyme-catalyzed reaction

cyanogen bromide

a reagent that cleaves proteins at internal methionine residues.

high-performance liquid chromatography (HPLC)

a sophisticated chromatography technique that gives fast and clean purifications.

cholesterol

a steroid that occurs in cell membranes; the precursor of other steroids.

antioxidant

a strong reducing agent, which is easily oxidized and thus prevents the oxidation of other substances.

inhibitor

a substance that decreases the rate of an enzyme-catalyzed reaction.

allosteric effector

a substance-substrate, inhibitor, or activator-that binds to an allosteric enzyme and affects its activity.

tandem mass spectrometry

a technique in which the output from one spectrometer is analyzed in a second spectrometer.

mass spectrometry (MS)

a technique that separates molecular fragments according to their mass-to-change ratio.

matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS)

a technique that uses a laser to ionize the protein sample for MS

western blot

a technique where proteins are first separated using gel electrophoresis and then transferred to a nitrocellulose membrane for analysis and identification.

gel-filtration chromatography

a type of column chromatography in which the molecules are separated according to size as they pass through the column

uncompetitive inhibition

a type of inhibition where the inhibitor can bind to ES, but not to free E

cation exchanger

a type of ion-exchange resin that has a net negative charge and binds to positively charged molecules flowing through the column.

anion exchanger

a type of ion-exchange resin that has a net positive charge and binds to negatively charged molecules flowing through it.

SN1

a uni-molecular nucleophilic substitution reaction; one of the most common types of organic reactions seen in biochemistry; the rate of the reaction follows first order kinetics.

heterotropic effects

allosteric effects that occur when different substances are bound to a protein.

homotropic effects

allosteric effects that occur when several identical molecules are bound to a protein.

protein chips

also called protein microarrays; small plates of a few centimeters on a side that can have tens thousands of proteins implanted.

lipid bilayer

an aggregate of a lipid molecule in which the polar head groups are in contact with water and the hydrophobic parts are not.

secondary antibody

an antibody that will react with the primary antibody used in an ELISA or western blot experiment

sequencer

an automated instrument used in determining the amino acid sequence of a peptide or the nucleotide sequence of a nucleic acid.

electrophile

an electron-poor substance that tends to react with centers of negative charge or polarization.

nucleophile

an electron-rich substance that tends to react with sites of positive charge or polarization.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

an electrophoretic technique that separates proteins on the basis of size.

ping-pong mechanism

an enzyme mechanism where a substrate binds to the enzyme and releases a product before the second substrate binds to the enzyme.

random mechanism

an enzyme mechanism where the substrates can bind to the enzyme in any order

ordered mechanism

an enzyme mechanism where the substrates have to bind to the enzyme in a specific order.

zymogen

an inactive protein that can be activated by specific hydrolysis of peptide bonds.

Beta carotene

an unsaturated hydrocarbon; the precursor of vitamin A

size-exclusion chromatography

another name for gel-filtration chromatography, which is a technique used to separate biomolecules based on size.

abzymes

antibodies that are produced against a transition-state analog and that have catalytic activity similar to that of a naturally occurring enzyme.

allosteric sites

binding sites on a target molecule for allosteric effectors.

enzymes

biological catalysts, usually globular proteins, with self-splicing RNA as the only exception.

V systems

combinations of allosteric enzymes and inhibitors or activators in which the presence of the inhibitor/activator changes the maximal velocity of the enzyme but not the substrate level that yields one-half Vmax.

K systems

combinations of allosteric enzymes and inhibitors or activators, in which the presence of the inhibitor/activator changes the substrate concentration that yields one-half Vmax.

transport proteins

components of a membrane that mediate the entry of specific substances into a cell.

sphingomyelins

compounds in which the primary alcohol of sphingosine is esterified to another amino alcohol.

irreversible inhibition

covalent binding of an inhibitor to an enzyme, causing permanent inactivation.

labeling

covalent modification of a specific residue on an enzyme.

first order

describes a reaction whose rate depends on the first power of the concentration of a single reactant.

second order

describes a reaction whose rate depends on the product of the concentrations of two reactants.

ELISA

enzyme-linked immunosorbent assay

free radicals

highly reactive molecules that have at least one unpaired electron.

stationary phase

in chromatography, the substance that selectively retards the flow of the sample, effecting the separation.

proton pumps

integral membrane proteins that create a hydrogen ion gradient across the membrane.

vitamin K

lipid soluble compound that plays an important role in blood clotting.

vitamin D

lipid soluble compound that regulates calcium and phosphorus metabolism.

ceramides

lipids that contain one fatty acid linked to sphingosine by an amide bond.

sphingolipids

lipids whose structure is based on sphingosine.

steroids

lipids with a characteristic fused-ring structure.

waxes

mixtures of esters of long chain carboxylic acids and long chain alcohols.

isozymes

multiple forms of an enzyme that catalyze the same overall reaction but have subtle physical and kinetic parameters.

-9, -6, -3

nM = 1 x 10^_____, uM = 1 x 10^____, mM = 1 x 10^____

coenzymes

non-protein substances that take part in enzymatic reactions and are regenerated at the end of the reaction.

native gel

one without SDS or another compound that would denature the proteins being separated

green chemistry

popular name for any chemistry that intentionally uses less toxic and more environmentally friendly chemicals.

peripheral proteins

proteins loosely bound to the outside of a membrane.

receptor proteins

proteins on a cell membrane with specific binding sites for extracellular substances.

nucleophilic substitution reactions

reactions in which one functional group is replaced by another as the result of nucleophilic attack.

amphipathic

refers to a molecule that has one end with a polar, water-soluble group and another end with a nonpolar hydrocarbon group that is insoluble in water.

zero order

refers to a reaction that proceeds at a constant rate, independent of the concentration of reactant.

liposomes

spherical aggregates of lipids arranged so that the polar head groups are in contact with water and the nonpolar tails are sequestered from water.

proteomics

study of interactions among all the proteins of the cell

leukotrienes

substances derived from leukocytes (white blood cells) that have 3 double bonds and are of pharmaceutical importance.

transition-state analogs

synthesized compounds that mimic the form of the transition state of an enzyme reaction.

TAMLs

synthetic molecules (terta-amido-macrocyclic ligand) that are used as detoxifiers

retinal

the aldehyde form of vitamin A

apoptosis

the biochemically and biologically programmed death of a cell.

steady state

the condition in which the concentration of an enzyme-substrate complex remains constant in spite of continuous turnover.

activation energy

the energy required to start a reaction.

active transport

the energy requiring process of moving substances into a cell against a concentration gradient.

sodium-potassium ion pump

the export of sodium ions from a cell with simultaneous inflow of potassium ions, both against concentration gradients.

transition state

the intermediate stage in a reaction in which old bonds break and new bonds are formed.

vitamin A

the lipid soluble compound responsible for the primary photochemical event in vision.

fluid-mosaic model

the model for membrane structure in which proteins and a lipid bilayer exist side by side without covalent bonds between the proteins and lipids.

alpha tocopherol

the most active form of vitamin E

turnover number

the number of moles of substrate that react per second per mole of enzyme

active site

the part of an enzyme to which the substrate binds and at which the reaction takes place.

passive transport

the process by which a substance enters a cell without an expenditure of energy by the cell.

feedback inhibition

the process by which the final product of a series of reactions inhibits the first reaction in the series.

homogenization

the process of breaking cells open to release the organelles.

catalysis

the process of increasing the rate of chemical reactions

simple diffusion

the process of passing through a pore or opening in a membrane without a requirement for a carrier or for the expenditure of energy.

K0.5

the substrate level at one-half Vmax in a K system.

proteome

the total protein content of the cell.