Chapter 7
What is a lysozyme?
An antibacterial enzyme in tears, saliva, and human milk. Active site is a long, deep cleft in the protein surface.
What is the relationship between k2 and k-1 when Kd = Km?
k2 << k-1
5 roles of all catalysts
1. Bind reactants 2. Weaken bonds of reactants 3. Increase local concentrations of reactants 4. Lower activation barrier for reaction 5. Release products
Most Km values range between:
10^-7 and 10^-1 M
What is the most targeted protein class of FDA approved drugs?
Enzymes at 304, next is transporters and GPCRs at 182 and 103, respectively.
Lyases
Enzymes that catalyze addition of groups to double bonds, or formation of double bonds by removal of groups.
1. In most enzymes, the active site consists of only a few residues. Why is the rest of the protein necessary?
The bulk of the protein is necessary to produce the correct tertiary structure for the substrate binding site and for proper orientation of the catalytic residues.
What are zymogens and what happens when activated?
An inactive substance which is converted into an enzyme when activated by another enzyme. Conversion of zymogen into a protease by cleavage of a single peptide bond switches on enzymatic activity, irreversibly.
How does structure change with phosphorylation?
Can induce major conformational changes
What is k1 limited by?
How fast molecules can diffuse in water: < 10^9 M-1s-1
Transferases
Enzymes that catalyze group transfer reactions
At relatively low concentrations of substrate, Vo _______ with an increase in _____
Increases almost linearly Increase in substrate concentration
What is the Michaelis constant?
Km, or the substrate concentration where half of the Vmax is observed. Therefore Km=Vmax/2. The lower the Km, the higher the affinity the enzyme has for the substrate
Kinase enzymes phosphorylate other proteins and enzymes. This is a common mechanism of covalently modifying enzymes in order to control their catalytic efficiency. Which of the following amino acids is not targeted for phosphorylation by kinases?
Leucine. Serine, threonine, and tyrosine are the three amino acids that are phosphorylated by kinase enzymes.
How does chymotrypsin bind into a catalytic group?
Preferred Phe, Trp, or Try fit into a slitlike hydrophobic pocket located near the catalytic groups.
How do enzymes usually bind to substrates?
With high affinity and specificity.
What happens to proteins in the absence of their metal cofactor?
They lose their native conformation.
Specificity pocket
A cavity on the enzyme surface at the active site that accommodates the residue on the N-terminal side of the scissile peptide bond
Gram-negative bacteria
Bacteria that have complex cell walls with less peptidoglycan but with lipopolysaccharides. Very toxic and hard to treat.
How do pKa residues vary?
Depending on chemical environment +/` 2-3 units
Noncompetitive inhibition
If Ki = Ki' the substrate binding to free enzyme is not affected.lots
Why isn't Km a true dissociation constant for ES binding? Except...
It depends on k2. Except if the dissociation rate k-1 is much faster than the catalytic rate k2, then: Km ~ k-1/k1 = Kd for the ES complex.
Catalytic perfection
Kcat/Km Max efficiency, approaches diffusion limited max (~10^8-1-^9 M-1 sec-1)
The lower the Km, the less ___ the enzyme requires, which gives ____
Less S Better catalysis
Most enzymes are ___ within a narrow pH range of ___
Optimally active 5-9
Aspartyl proteases
Pepsin, HIV protease
T or F: enzymes can be easily stereospecific.
True
Crixivan
A protease inhibitor used as a component of highly active antiretroviral therapy to treat HIV/AIDS.
Proteases often operate by...
Forming a transient covalent intermediate with part of the substrate, then cleaving it and regaining the free active site.
Metalloproteases
Thermolysin, matrix metalloproteinases
Lock and key vs. induced fit enzyme specificity
1. Lock and key: substrate binds to the enzyme perfectly. 2. Induced fit: enzyme is flexible to accommodate the ill fitting substrate. Permits a much larger number of weaker interactions between substrate and enzyme.
Ligases
Enzymes that catalyze formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to ATP cleavage.
How do lysozyme interact with its substrate?
Lysozyme is thought to strain ring D by forcing it to a half-chair conformation.
Saturation effect
Observed in enzyme catalysis when plotting the initial velocity Vo against the substrate concentration.
What happens to a protease/peptide when kcat/Km is high, medium, and low?
Same thing. High catalytic efficiency means the enzyme binds and carries out catalysis immediately.
A mutant of chymotrypsin is constructed in which a neutral side chain at the bottom of the specificity pocket is changed to Asp. What effect do you predict for this Ser189->Asp189 mutant?
****
4 Major factors in catalysis
1. Entropy reduction or approximation 2. General acid-bases as active agents 3. Transient covalent bond formation 4. Metal ions
Cysteine proteases
Caspases
Reversible inhibition and 3 classes
Noncovalent binding of small biomolecules or proteins to the enzyme subunit. -Can be decreased by diluting the enzyme reaction 3 types: competitive, uncompetitive, mixed.
____ are needed to regulate proteolysis
Inhibitors. Inhibitor mimics the substrate, but binds very tightly, rarely progressing to the transition state and is turned over slowly (several months) Kd ~ 10^-13 M Similarity in free and bound forms
What amino acids are most frequently phosphorylated? Methylated/acetylated?
Ser, Thr, and Tyr Lysines
Graph of ATCase
Sigmoidal. Not amenable to analysis by M-M kinetics.
In acid/base H bonding, what does the dashed bond represent?
The direction of proton transfer from the proton donor to the proton acceptor (wide end of the bond near the proton acceptor)
In the Michaelis-Menten equation, if we double the enzyme concentration, ____ doubles, but ____ does not change.
Vmax doubles Km does not change
2. If you wanted to improve the catalytic efficiency of an enzyme, would you mutate amino acid residues to increase binding affinity for the substrate or increase the binding of the transition state?
You make the transition state bind more tightly in order to lower the activation energy of the reaction. Binding the substrate more tightly would make a higher activation energy.
Example of covalent inhibition of enzyme activity
DIPF biding to Ser195
Enzyme bioavailability is controlled by...
Biochemical processes involved in protein synthesis and localization (grey arrows)
The following figure shows one step in the chymotrypsin reaction mechanism. Place the labels on the appropriate circles, which refer to the species they are closest to in the figure.
Both amino acids Asp102 (D) and His57 (B) are parts of the catalytic triad in the binding pocket of chymotrypsin and are involved in its proteolytic activity. The substrate (C) is cleaved due to the formation of two tetrahedral intermediates, with (A) indicating the first intermediate. This is facilitated by the hydrogen-bond network of the amino acids in the binding pocket of chymotrypsin.
Place the labels in the appropriate locations on the two graphs.
Graph A, which shows the linear part of experimental data, tracks the rate of product formation versus time of the reaction with increasing concentrations of substrate. The more substrate added, the faster the product is formed (up to a certain point). These data are then transposed to the Michaelis-Menten graph (Graph B), in which the initial velocity (the slope of the lines in Graph A) is plotted on the y-axis, and the initial substrate concentration on the x-axis. The greater the substrate concentration, the greater the initial velocity in Graph B, up to a point termed the maximum velocity of the reaction.
Which of the following is not an assumption that is made when applying Michaelis-Menten kinetics to an enzyme?
The rate constant of E + P re-associating to form the ES complex must be considered. Three simplifying assumptions are made about reaction conditions when applying Michaelis-Menten kinetics to an enzyme reaction: (1) no appreciable product has yet been formed, (2) product release is a rapid step in the reaction, and (3) steady-state conditions are reached quickly. The rate constant of the reverse reaction, in which E + P re-associates to form the ES complex, is assumed to be zero, and need not be considered.
__________ are stable molecules that mimic a transition state and bind to the ___________ of enzymes. The ________ affinity of these analogs for the enzymes supports the transition state theory because it is assumed that the similarly shaped transition state is energetically favorable.
Transition state analogs Active site High If a transition state analog has a very high affinity for the active site of an enzyme, and that stable analog is designed to mimic the proposed transition state of that enzyme mechanism, then that lends support to the transition state theory of that enzyme mechanism.
T or F: Allosterically regulated enzymes do not follow M-M kinetics.
True. ATCase shows cooperatively (sigmoidal curve). The binding of substrate to one active site increases the activity at the other active sites.
12.An enzyme has a single active site at which it can bind and hydrolyze either X or Y; however, the enzyme cannot bind X and Y at the same time. Answer the following questions regarding the Km and vmax of this enzyme. a) Will the Km for X be affected if Y is present in the reaction mixture? Explain. b) Will vmax for X be affected if Y is present in the reaction mixture? Explain. c) Is it possible for vmax and vmax/Km to show a different dependence on pH? Explain.
a) Yes, Y is a competitive inhibitor of X; the apparent Km for X will increase. b) No, the vmax for X will be unaffected. c) Yes, the pH dependence of vmax reflects the ionization of catalytic site residues, whereas pH dependence of vmax/Km will reflect the ionization state of substrate binding residues.
Reaction rates exhibit ____ curves in dependence of ___
bell-shaped pH
Which of the following statements is false, considering Michaelis-Menten enzyme behavior and plots?
kcat is the turnover number of an enzyme-catalyzed reaction. Decreasing the concentration of an enzyme will result in a decrease of Vmax, but will not change the Km value of that reaction. The Michaelis-Menten constant is a constant that is specific for a particular enzyme.
Covalent catalysis
Accelerates reaction rates through the transient formation of a catalyst-substrate covalent bond. Covalent bond provides chemistry that speeds the reaction
What are active sites?
Binding pockets in the enzyme that bind to the substrate and promote catalytic reactions.
Proteases are studied with _____ ester substances
Chromogenic Chromogenic substrates are peptides that react with proteolytic enzymes and are converted to insoluble, colored products that precipitate onto the membrane.
Nucleophile role in covalent catalysis
Nucleophilic group on enzyme attacks an electrophilic group on the substrate = nucleophilic catalysis
What does catalytic efficiency define?
The efficiency of the enzyme. -Higher values mean more efficiency.
How does esterolysis proceed? What is kcat, Km, rate enhancement?
Through a 2-step acyl-oxygen fission that includes a high energy anionic tetrahedral intermediate. Kcat: 5.5 min-1 Km: 391 uM Rate enhancement: 1.6 x 10^4
5 reasons why phosphorylation is used to regulate enzyme activity
1. Rapidly reversible. Can quickly switch between active and inactive enzyme forms 2. Relatively inexpensive; no new protein synthesis needed 3. Results in large delta Grxn; can shift conformational equilibrium by factor of 10^4 4. Timing can be adjusted to fit physiological needs of cell 5. Effects can be rapidly amplified via kinase cascade
8 steps of peptide hydrolysis by chymotrypsin
1. Substrate binding 2. Nucleophilic attack of serine on peptide carbonyl group (forming tetrahedral intermediate) 3. Collapse of tetrahedral intermediate (acyl-enzyme formed) 4.Release of the amine component 5. Water binding 6. Nucleophilic attack of water on acyl-enzyme intermediate (forming tetrahedral intermediate) 7. Collapse of the tetrahedral intermediate 8. Release of the carboxylic acid component. The dashed green lines represent hydrogen bonds.
Michaelis-Menten Kinetics
A kinetic pattern in which the initial rate of an enzyme-catalyzed reaction exhibits a hyperbolic dependence on substrate concentration. Need to bind first, then carry out a reaction.
The following figure shows the activity curves of the enzyme ATCase, with and without allosteric effector molecules bound to the enzyme. ATCase catalyzes the formation of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate. "Control" in this figure refers to ATCase in which no allosteric effector molecule is bound. Label the curves as they correspond to the descriptions. Addition of CTPAddition of ATPControl
ATP stimulates ATCase activity so that the initial velocity of the reaction will be higher at lower aspartate concentrations (B). CTP, however, inhibits ATCase activity such that more aspartate is needed to reach similar reaction velocities (A). The control (C) indicates that no allosteric regulator has been added to the reaction.
What is the steady-state of the Michaelis complex ES?
At steady state, the production and consumption of the transition state proceeds at the same rate. The concentration of ES is constant.
4. It was found that for the reaction A <-> B, the forward rate constant kF in the absence of enzyme was 1E-2 s-1, whereas the kF in the presence of enzyme was 5E6 s-1. b) If the equilibrium constant Keq for the A <-> B reaction is 1E3 in the absence of enzyme, what is the Key in the presence of enzyme? Explain.
Catalysts do not change the Keq, so the Keq remains 1E3.
How does luciferase work?
Catalyzes the conversion of luciferin to oxyluciferin with a photon as a byproduct. 1. Formation of acid anhydride between carboxylic group of D-firefly luciferin and AMP. 2. Oxygenation follows, w/ release of AMP group and formation of short lived peroxide the cyclizes into dioxetanone structure, which collapses releasing a Co2 molecule.
What happens when substrate binds to the active site?
Change is induced in the enzyme.
What happens when chymotrypsinogen is cleaved?
Chymotrypsinogen is an inactive precursor (zymogen) of chymotrypsin, a digestive enzyme which breaks proteins down into smaller peptides. It is activated into its active form by another enzyme called trypsin. This active form is called π-chymotrypsin and is used to create α-chymotrypsin.
4. Describe the functional roles of enzyme cofactors.
Cofactors are small molecules that aid in catalytic reaction mechanisms by providing additional chemical groups to supplement the chemistry of amino acid functional groups. These include inorganic ions such as Fe2+, Mg2+, Mn2+, Cu2+, and Zn2+, or coenzymes which are inorganic compounds. Coenzymes can be loosely or tightly associated with enzymes, or even covalently attached. Most vitamins from nutritional sources are coenzymes. Coenzymes that are permanently associated with enzymes, such as the heme of catalase, are called prosthetic groups.
Decomposition of the tetrahedral intermediate
Decomposition to the acyl-enzyme intermediate and scission of the peptide bond Driven by donation of proton from N3 of His57 (general acid catalysis). Helped by polarizing effect of Asp 102 on His 57 (electrostatic catalysis)f
What are isozymes?
Enzymes that differ in amino acid sequence that catalyze the same reaction. -Different kinetic parameters or respond to different regulatory molecules -Encoded by different genes -Permits fine-tuning of metabolism to meet needs of given tissue or developmental stage.
What does clot formation involve?
A zymogen-enzyme cascade
6. How does the addition of an enzyme to a chemical reaction affect each of the following parameters (no affect, increase, or decrease)? a) Standard free energy of the reaction b) Activation energy of the reaction c) Initial velocity of the reaction d) Equilibrium constant of the reaction e) Time to reach equilibrium
a) no effect b) decrease c) increase d) no effect e) decrease
What roles do ATP and Mg2+ play in hexokinase activity?
ATP and Mg2+ are both cofactors (a non-protein chemical compound that is required for the protein's biological activity).
NAG-NAM in bacterial cell walls
Alternating polysaccharide component of bacterial cell walls. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).
Function of enzyme alcohol dehydrogenase
Breaks down ethanol (toxic) to acetaldehyde (very toxic) which is quickly converted to nontoxic acetate.
How do proteases operate?
By forming a transient covalent intermediate with part of the substrate, then cleaving it and regaining the free active site.
How can competitive inhibition be overcome?
By increasing concentration of substrate. Vmax is unchanged, but Km increases
3. How do enzymes increase rates of biochemical reactions?
By lowering the activation energy. They provide favorable conditions to lower the transition energy barrier, which increases the rate of the reaction without altering overall delta G.
Which of the following is false regarding the protein pepsin?
Pepsinogen is the active form of pepsin. Pepsinogen is the inactive precursor of pepsin. When in an acidic environment, like the pH of the stomach, pepsinogen self cleaves to form the active pepsin protein. Pepsin is a protease that hydrolyzes peptide bonds, aiding in digestion of food.
What does the graph look like when the binding affinity for the enzyme-substrate complex is greater (Ki > Ki')
The apparent affinity for substrate increases (and Km decreases)
How to catalysts and enzymes make reactions run quicker?
They stabilize the transition state.
Two strategies for stabilization of charged transition states
1. Electrostatic interactions with active site amino acids and/or metal ions. 2. Hydrogen bonds with general acids or bases
Graph of reaction velocity versus concentration of substrate when reaching a maximum-- what is the significance of reaching maximal velocity?
A discrete ES complex is being formed
Why does the enzyme distort the D ring?
Better alignment of orbitals leading to cleavage
Which of the following is not a biochemical process affecting the bioavailability of enzymes?
Binding of a competitive inhibitor. RNA synthesis and processing as well as protein synthesis and degradation all affect the bioavailability of enzymes. The binding of a competitive inhibitor to an enzyme, however, affects the catalytic efficiency of the enzyme, but not its bioavailability.
Structure of remdesivir and how it works
Blocks enzyme required for viral replication
What happens to Km and Vmax during uncompetitive inhibition?
Both Km and Vmax are decreased. Uncompetitive inhibitors bind to enzyme substrate complexes and alter the active site conformation
2. Describe two ways in which enzymes are regulated.
By increases or decreases in enzyme levels (bioavailability) and by changes in catalytic efficiency. Bioavailability can be at the level of gene expression or protein synthesis, whereas catalytic efficiency is regulated by post translational modifications or binding of allosteric regulators.
Sort the following statements into the correct bins based on whether they most appropriately describe the binding pocket of chymotrypsin, trypsin, or elastase.
Chymotrypsin, trypsin, and elastase are all serine proteases that bind different types of amino acids due to the different structures of their binding pockets. The serine and glycines of chymotrypsin, along with its relatively large size, allow larger aromatic amino acids to bind. The negatively charged binding pocket of trypsin (with aspartic acid and two glycines) allows positively charged amino acids such as arginine to enter it, while the small size of the elastase binding pocket (with threonine, valine, and serine) allows only small amino acids to enter it.
The catalytic triad
Ser, His, Asp
Role of sticks, an arbitrary (imaginary) enzyme
Shows that before product (metal rod) is released, it must first be bent (transition state)
7. Briefly describe the two phases of the proposed mechanism for the chymotrypsin reaction.
First, a covalent acyl-enzyme intermediate is formed between Ser195 and the polypeptide substrate, to promote cleavage of the scissile peptide bond and release the amino acid terminal polypeptide fragment. Second, the enzyme is regenerated in a series of steps resulting in deacylation and release of the carboxyl-terminal polypeptide fragment.
How did Pauling propose engineering proteins to stabilize a transition state?
Generate antibodies to stable analogs of the transition state.
What are lysozyme's catalytic residues?
Glu 35 (pKa = 6)and Asp 52 (pKa = 4); optimal pH around 5 (Two carboxylate side chains involved in catalysis)
What is the vitamin source of NAD+/NADH?
Vitamin B3 (niacin). Representative enzyme: lactate dehydrogenase Role in catalysis: Oxidation-reduction
Enzyme specificity: Chymotrypsin, Trypsin, Elastase
Chymotrypsin: Large substrate binding pocket accommodates aromatic residues such as tyrosine. Trypsin: Binding pocket with a negatively charged residue at the bottom accommodates residues with a positive charge such as arginine Elastase: The and Val residues close off the binding pocket so that only small residues such as alanine can be accommodated
How is Trypsin similar in binding to chymotrypsin?
Trypsin recognizes Lys and Arg in a deep pocket similar to that of chymotrypsin
Irreversible inhibition
-Inhibitory molecule forms a covalent bond or very strong non covalent bond with catalytic groups in the enzyme active site -"Kill" the enzyme by tight binding to the enzyme
Enzymes only alter the _____, and not the _____.
Reaction rate NOT reaction equilibrium
9. What are the three classes of reversible inhibitors? Briefly describe them and their effects on apparent Km and vmax.
1. A competitive inhibitor competes with the substrate for binding to the active site. This shifts the apparent Km value for the enzyme to the right as a function of increasing inhibitor concentration, but does not change the vmax of the rxn. 2. Uncompetitive inhibitors bind to enzyme-substrate complies (ES) and alter the active site conformation, reducing catalytic activity, and resulting in a decrease in the Km and vmax without altering the Km/vmax ratio. 3. Mixed inhibitors also bind to sites distinct from the active site, but they bind to both the enzyme and the enzyme-substrate complex. A mixed inhibitor decreases vmax and may increase or decrease Km, depending on the relative KI and KI' values.
5 ways cells regulate enzyme activity
1. Allosteric proteins: enzymes have a catalytic site and separate regulatory sites responsive to signaling molecules 2. Isozymes: Multiple forms of kinetically distinct enzymes in different tissues or organelles 3. Covalent modification: Phosphorylation, acetylation, etc. 4. Proteolytic activation: Zymogens and clotting cascade 5. Control of enzyme biosynthesis via gene expression.
5. In what three ways do enzymes lower the activation energy of a reaction?
1. By providing an alternative path to product formation, like forming stable reaction intermediates that are covalently attached to the enzyme. 2. By lowering the activation barrier through stabilization of the transition state. 3. By increasing the substrate's ground state energy by limiting bond rotation within the active site, lowering the energy difference between the ground state and transition state.
Catalytically important residues at pH 4/5, 6-7. 10
4/5: Glu, Asp 6-7: His 10: Lys
6. Describe the structure and function of the catalytic triad in chymotrypsin.
A set of three amino acids that form a hydrogen-bonded network required for catalysis. In chymotrypsin, these three amino acids are Ser195, His57, and Asp102.
Label the different energies on the following energy diagram. On this graph, the x-axis is the reaction coordinate, while the y-axis is energy.
Adding a catalyst to a reaction can stabilize the transition state, thereby reducing the activation energy of that reaction. However, the overall free energy of the reaction remains the same with or without a catalyst.
Gram-positive bacteria
Bacteria that have a thick peptido glycan cell wall, and no outer membrane. They stain very darkly (purple) in Gram stain.
What happens to Km and vmax for mixed inhibition?
Can bind to both the enzyme and the enzyme-substrate complex. Lowers vmax and may increase or decrease Km
Pepsin is an enzyme used to help digest food in the stomach by cleaving peptide bonds. Incubating pepsinogen in a stomach whose pH has been artificially increased to 7.4 would ____ the rate at which food can be digested.
Decrease. Pepsinogen must be cleaved to form the active pepsin, which is the enzyme that cleaves the peptide bonds in food to aid digestion. This cleavage occurs best in the acidic pH of the stomach, a pH of approximately 1.5. Increasing the pH to 7.4 would significantly reduce the amount of pepsin produced, so the rate of food digestion would decrease.
Large substrate concentration (S >> E) will saturate binding sites and the reaction will...
Go at a maximal rate
There are three primary types of reactions used in metabolite transformation, listed in the bins. Sort the statements into the correct bins, depending on the type of reaction to which they are most applicable.
Isomerization reactions do not change the molecular formula of the reactant when forming product; they are intramolecular rearrangements. This is the same as saying the product has the same number and identity of atoms as the reactants. Condensation reactions combine two reactants together to form a larger product, usually by losing a water molecule. Hydrolysis or dehydration reactions cleave a substrate into two products by adding or removing water. Triose phosphate isomerase, prenyl transferase, and chymotrypsin are examples of enzymes that perform isomerization, condensation, and hydrolysis, respectively.
Catalytic efficiency is determined by...
Protein modifications (teal arrows)
The GAC is a ____ and the GBC is a ______.
Proton donor Proton acceptor
General Acid-Base catalysis
Proton transfer from an acid lowers the free energy of a reaction's transition and state and speeds up the reaction.
4. It was found that for the reaction A <-> B, the forward rate constant kF in the absence of enzyme was 1E-2 s-1, whereas the kF in the presence of enzyme was 5E6 s-1. a) Calculate the rate enhancement provided by the enzyme for this chemical reaction.
Rate enhancement = kF catalyzed / kF uncatalyzed = 5E6 s-1 / 1E-2 s-1 = 5E8 fold enhancement
2 kinds of subunits within allosteric enzymes
Regulatory (r) subunits and catalytic (c) subunits
What is the final product of ATCase?
Structurally unrelated to the substrates or product of ATCase.
What does the graph look like if binding affinity of the inhibitor for the enzyme alone is greater (Ki'>Ki)
The affinity for the substrate decreases (which means Km increases)
Label the parts of the Michaelis-Menten graph.(Note: The pink line represents the enzyme reaction with a high concentration of enzyme, while the green line represents the same enzyme reaction with a lower enzyme concentration.)
The plateau region of the graph is the vmax, and half of that value on the y-axis is 1/2vmax. The Km is the value on the x-axis that corresponds to 1/2vmax.
Primary structure of HEW lysozyme
The primary structure of lysozyme is a single polypeptide containing 129 amino acids. In physiological conditions, lysozyme is folded into a compact, globular structure with a long cleft in the protein surface.
Which of the following is/are true regarding observed effects of pH on enzyme activity and rate, and the underlying causes of these effects?
The rate of an enzyme-catalyzed reaction is maximized at the optimal pH for that enzyme. At this pH, the active site is in the correct orientation for substrate binding and product formation. The optimal pH will vary for different enzymes, but outside of the narrow optimized pH range, the activity and reaction rate of the enzyme will usually drop precipitously.
7. A mutation in the active site of an enzyme results in a large increase in stabilization of the ES complex, while there is no change in the stabilization of the transition state complex. What effect will this have on the rate of product formation?
This would decrease rate of product formation because stabilization of ES increases the activation energy (even though the transition state energy is unchanged).
How do proteases show burst phase kinetics?
Upon adding enzyme to substrate, a large initial velocity is exhibited that levels off once all enzymes have been saturated. At this point enzyme velocity linearly increases. The initial high velocity is called the burst phase.
Example of steady state concept
Water flows into a sink and flows out of a drain. The rate at which water flows out of the drain is proportional to the pressure of the water, which depends on how much water is in the sink. As more water accumulates in the sink, a steady state is reached when the flow in from the faucet is matched by the flow out through the drain. The amount of water in the sink is constant under these conditions. Compare the buildup of water in the sink with the increase of the concentration of B as saturation reaches its peak in the previous graphs.
Catalysis can involve both ____ and _____
proximity, orientation
2 modes of enzyme regulation
1. Bioavailability 2. Catalytic efficiency
By oriented binding and immobilization of the substrate, enzymes facilitate catalysis in three ways:
1. Bring substrates close to catalytic residues 2. Binding of substrate in proper orientation 3. Freezing out of translational and rotational mobility of the substrate
How does proteolytic cleavage differ from phosphorylation?
1. Can occur outside of cells, since ATP not needed to convert zymogen into active form of enzyme 2. Not reversible. Inactivation of active enzyme must occur by either degradation of enzyme or by inhibition (e.g. due to binding of inhibitory protein to active enzyme)
Oxyanion hole
A region on certain proteolytic enzymes that stabilizes the oxyanion constituent of the tetrahedral intermediate of the reaction.
HIV protease
An enzyme that converts immature, noninfectious HIV to its infectious state within the cell
10. Identify the two chemical structures that represent the first and second tetrahedral intermediates in the chymotrypsin-catalyzed cleavage of a peptide.
C is the first intermediate and E is the second intermediate.
What is an example of some enzymes being more specific than others?
Consider proteolytic enzymes (which degrade proteins by breaking amide bonds). 1. Papain: displays no specific protein sequences and cleaves at all amide bonds. 2. Thrombin: cleaves only between Arg-Gly bonds in certain recognition sequences
8. Explain how changes in pH can affect enzyme activity.
Eight amino acids have ionizable side chains (can gain or lose a proton, depending on the local environment and effective pKa). Changes in pH alter active-site chemistry by protonation and deprotonation of side chains, disrupting critical tertiary structures within the protein, altering enzyme activity
ATCase allosteric inhibition
Example of feedback inhibition. Carbamoyl phosphate combines with Asp to give N-carbamoyl aspartate, which goes on in about 6 more steps to form rUTP and finally CTP, precursors of RNA and DNA rCTP inhibits ATCase
How does the graph of burst phase kinetics show proof of a 2 step mechanism?
First fast step, slower second step. Burst phase is part one.
Inter vs. intra-molecular reactions
In intermolecular reactions, covalency changes take place in two separate molecules; in intramolecular reactions, two or more reaction sites within the same molecule are involved.
What mechanism does hexokinase exhibit?
Induced fit.
Sarin gas is a nerve gas that is considered a weapon of mass destruction. The use, production, and stockpiling of chemical weapons has been banned by international treaty due to the extremely painful death they cause. Nonetheless, some countries have stockpiles of sarin gas, which has been used on human populations on occasion. Sarin gas poisoning is difficult to treat because sarin forms a covalent bond with acetylcholinesterase, making it a/an _______ inhibitor.
Irreversible. Since sarin gas forms a covalent bond with its enzyme target, acetylcholinesterase, it is an irreversible inhibitor.
Turnover number
Maximum number of substrate molecules an enzyme molecule converts to product each second. k2 = catalytic rate constant (kcat) Enzyme with maximal k2 is best
The characteristics of the active site microenvironment of an enzyme can be largely independent of individual catalytic mechanisms. In general, active site microenvironments promote catalysis in which of the following ways?
The active sites of enzymes increase the rate of reaction because they decrease the activation energy of the reaction, and the physical microenvironment provides an optimal orientation of the substrate relative to reactive functional groups while excluding excess solvent, such as water. Although some active sites may have amino acids that form salt bridges with the amino acids from a substrate, not all do, so this is not a generic strategy of active site microenvironments.
The enzyme ATCase is regulated by allosteric mechanisms. The binding of ATP to ATCase _______ the activity of ATCase, while the binding of CTP _________ the activity of ATCase. When _________ binds to ATCase, the R state conformation of ATCase forms, causing the catalytic site to become
Upregulates Downregulates ATP Activated The enzyme ATCase is regulated by allosteric mechanisms. The binding of molecule ATP to ATCase upregulates the activity of ATCase, while the binding of molecule CTP downregulates the activity of ATCase. When ATP binds to ATCase, the R state conformation of ATCase forms, causing the catalytic site to become active.
10. What is the mechanism of feedback inhibition?
Feedback inhibition occurs when the end product of a pathway functions as an inhibitor of the first enzyme in the pathway.
What rate constant limits catalytic perfection?
- The ultimate limit on the value of kcat/Km is set by k1, the rate of formation of the ES complex. -The perfect enzyme has kcat/Km as close to k1 as possible! -If the ratio is ~k1, every collision between E and S would be productive.
Extracellular signal-regulated kinase (ERK) signaling
A chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell
Which of the following Lineweaver-Burk plots shows the effects of a noncompetitive inhibitor on an enzyme-catalyzed reaction?(Note: The yellow line indicates no inhibitor, the green line indicates a low inhibitor concentration, and the pink line indicates a high inhibitor concentration.)
A noncompetitive inhibitor decreases the vmax of a reaction without affecting the Km value. Therefore, on a Lineweaver-Burk plot, increasing concentrations of a noncompetitive inhibitor will increase the slope of the line and the value of the y-axis intercept, while leaving the x-axis intercept unchanged.
Forming the acyl enzyme intermediate leads too...
A push-pull overall mechanism. -There are 2 sets of tetrahedral intermediates. -Specificity is controlled by a separate pocket in the enzyme adjacent to the triad.
How do cysteine, aspartyl, and metalloproteases proceed?
All via a tetrahedral intermediate
Sort the following statements into the appropriate bin, considering if they apply to competitive, uncompetitive, or mixed inhibitors.
Competitive inhibitors bind to the active site of an enzyme, they do not change the vmax of the reaction, and their action can be negated by increasing the concentration of substrate. Uncompetitive inhibitors do not bind to the active site or the free enzyme, and they decrease both the vmax and the Km of the reaction. Mixed inhibitors do not bind to the active site of an enzyme either, but they can bind both the free enzyme and the enzyme-substrate (ES) complex.
Isomerases
Enzymes that catalyze transfer or groups within molecules to yield isomeric forms.
How does catalysis by hexokinase work?
Hexokinase catalyzes glycolysis with ATP-dependent phosphorylation of glucose.
Which of these mutations is the most likely to eliminate the ability of chymotrypsin to perform its function?
His57→Tyr Although any of the four amino acid changes listed in the catalytic triad very well may eliminate the function of chymotrypsin, mutating histidine to tyrosine is the most likely to do so. Even though this mutation maintains a ring structure and a functional group, the hydrogen-bonding capability of histidine will be partially lost. In the triad, histidine at position 57 serves as both a hydrogen-bond donor to Asp102 and a hydrogen-bond acceptor to Ser195. The hydrogen-bonding network between the three amino acids in the triad is essential for catalytic function. Tyrosine is only capable of one hydrogen bond, being a hydrogen-bond donor, whereas the native histidine forms two hydrogen bonds with neighboring amino acid R groups.
Many over-the-counter painkillers and anti-inflammatory drugs, such as aspirin and ibuprofen, function by limiting the amount of prostaglandins the body produces. Prostaglandins go to an area of injury or infection and bring with them redness, swelling, and pain. If the amount of prostaglandins in a person is reduced, then the pain and swelling will be reduced. In order to reduce the amount of prostaglandins produced, pain relievers interrupt the prostaglandin biosynthetic pathway by binding to an essential enzyme in this pathway called cyclooxygenase (COX). When a painkiller binds to COX, the enzyme can no longer catalyze its associated reaction. This fact means that aspirin and ibuprofen are examples of ________.
Inhibitors. Aspirin and ibuprofen are both inhibitors because they bind to the enzyme COX and reduce or eliminate its ability to function as a catalyst.
Initial velocity
Measured at the beginning of the enzyme catalyzed reaction, when substrate concentration can be considered constant, will decrease as the reaction progresses
How does cigarette smoking oxidize Met 358 of alpha1-antitrypsin?
Met 358 is crucial for high affinity binding of a1-antitrypsin to the active site of elastase. Cigarette smoking oxidizes Met 358, decreasing its affinity for elastase. The loss of control of elastase activity results in breakdown of alveolar cell walls in lung.
Experiments are performed to determine the initial reaction velocity of an enzyme-catalyzed reaction. What is NOT held constant so that the initial velocities can be used to plot the Michaelis-Menten graph?
Substrate concentration. Experiments are performed to determine the initial reaction velocity of an enzyme-catalyzed reaction. The initial velocities can be used to plot the Michaelis-Menten graph. The substrate concentration is NOT held constant. The substrate concentration is varied as this is plotted against the initial velocity of the reactions to create the Michaelis-Menten plot. Temperature, enzyme concentration, and the volume of the reaction are all held constant in these reactions, however.
Label the following figure. (Not all labels may be used.) Remember that an enzyme's active site binds to substrate based on the shape of both the enzyme and active site, as well as the attraction between amino acid residues (or cofactors) in the active site and on the substrate. For example, opposite charges will attract one another, and nonpolar residues will attract one another.
This enzyme is using the induced-fit model of substrate binding, where the shape of the enzyme active site changes after substrate binding. The substrate binding, and thus shape change of the active site, is facilitated in part by charged residues in the active site being attracted to oppositely charged residues on the substrate(s), and hydrophobic regions in the active site being attracted to hydrophobic residues of the substrate(s). Once product is formed and no substrates are bound to the enzyme active site, the enzyme returns to its original conformation.
Are catalytic triads found in other hydrolytic enzymes?
Yes. Catalytic triads are most commonly found in hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, acylases, lipases and β-lactamases).
What is the rate influenced by when S is low vs. S is high?
a) S is low: rate influenced by S b) S is high: rate determined by Et,k2
What is elastase inhibited by?
alpha1-antitrypsin Inhibits elastase, a serine protease that is responsible for remodeling collagen.
What is the relationship between Km and Kd at rapid equilibrium?
Km = Kd
Structure-based drug design strategies often devise competitive inhibitors that bind to certain enzyme active sites. For example, saquinavir and indinavir are designed HIV protease inhibitors that bind to the aspartate protease enzyme of HIV, which is required for the virus to produce functional proteins for further HIV infection. Which of the following statements are true about saquinavir and indinavir?
-Very high local concentrations of proteins with Phe-Pro or Tyr-Pro peptide bonds would reduce the effectiveness of saquinavir and indinavir in limiting HIV's infectivity of new cells. -Very high local concentrations of proteins with Phe-Pro or Tyr-Pro peptide bonds would reduce the effectiveness of saquinavir and indinavir in limiting HIV's infectivity of new cells. -Very high local concentrations of proteins with Phe-Pro or Tyr-Pro peptide bonds would reduce the effectiveness of saquinavir and indinavir in limiting HIV's infectivity of new cells.
Hydrolases
Enzymes that catalyze hydrolysis reactions (transfer of functional groups to water)
Oxidoreductases
Enzymes that catalyze transfer of electrons (hydride ions or H atoms)
9. The activity of chymotrypsin changes as the pH changes in the range of pH 5-9, as shown on the following graph. From your understanding of the chemical mechanism of the chymotrypsin reaction, explain the pH effect on chymotrypsin activity by answering parts a, b, and c. a) What amino acid functional group on which residue is likely responsible for the effect of pH? b) What is the apparent pKa of that functional group? Indicate on the graph how you deduced the pKa. c) Briefly explain in terms of the chymotrypsin mechanism why the activity increases as the pH increases in this pH range.
a) The imidazole of the active site His residue. b) The pH at which the concentration of base = concentration of acid, so protein would be 50% active at pH 6.8. To see this on the graph, draw a horizontal line from the 50 mark on y axis the vertical line down where this meets the line. c) In the first catalytic step of the mechanism, the active site His is unprotonated because it acts as a general base to accept a proton from the active site Ser.
