Biochemistry 1
Reaction mechanisms in the enzyme pocket?
They are entirely a function of the chemistry of the -R groups found in that pocket. You can usually predict accurately the amino acids present in a protein ligand or in an enzyme active site, just by knowing how the protein is functioning (at the very least, you can narrow it down to a select group of possibilities).
immune system
antigens, antibodies
Will a substrate bind in an active site?
The answer depends on the existence of complementary charges on the -R groups, and/or the hydrophilicity or hydrophobicity of the -R groups.
Coenzymes
Non-protein species NOT permanently attached to the enzyme but required by the enzyme to function.
Prosthetic groups
Non-protein species that ARE permanently attached to the enzyme and are required by the enzyme to function.
Kinesins
Move along microtubules from (+) to (-) [ center of cell to periphery; nerve cell body -> dendrite].
Dyneins
Move along microtubules from (+) to (-) [ periphery to center of the cell; nerve cell dendrite -> cell body].
Proceeding from acidic to basic (Low to High pH)
1) a-COOH group => pKa = 2 2) -R group, ACIDIC => pKa = 4 (Asp=3.7; Glu=4.5) 3) -R group, His => pKa= 6 4) a-NH3+ group => pKa= 9 5) -R group, BASIC => pKa = 11-12 (Lys=10.7; Arg=12)
Acid- Base Functionality of Amino Acids
Amino acids are WEAK acids. Each amino acid has a MINIMUM of two acidic protons: -COOH and -NH3+ Some amino acids have acidic side chains, and therefore three acidic protons. Per the above statements, each amino acid has either two or three pKa values. When the pH is near the pKa of one of the acidic protons the amino acid acts as as buffer.
Draw a fischer projection of the amino acid alanine in both its L- and D-forms.
A Fischer projection is a representation of a three dimensional molecule drawn in two dimensions. A tetrahedral carbon is represented as two crossed lines, and the groups attached to that carbon are displayed. The horizontal line is extending "out" of the paper, toward you, and the vertical line is behind the plane of the paper, away from you. Because of this, Fischer projections can be rotated 180° but not 90° or 270°. 180° rotation just flips the molecule over: the same R groups are extending forward or backward. But if you rotate the molecule just 90° in either direction, you have changed which R groups are above or below the plane of the paper, which changes the stereochemistry of the molecule. D - and L- amino acids are mirror images of one another, and they are not identical compounds. Think of your left and right hands. They are mirror images, but you cannot superimpose one upon the other because they are arranged in a fundamentally different way. L - amino acids are predominant in nature, although a few D - amino acids are used by some bacteria.
Provide a real-life human body example of a reaction that requires a) a coenzyme, and b) a prosthetic group. Draw and describe the function/ role of the cofactor in the reaction.
A coenzyme is an organic or organometallic cofactor that is required by an enzyme to function properly. Prosthetic groups are coenzymes that are covalently linked (or non-covalently but very tightly bound) to their enzyme partner. Nicotinamide adenine dinucleotide (NAD) is a common coenzyme in the human body. It functions to transfer electrons from one molecule to another in redox reactions, carried out by oxidoreductases. An example of this is complex I of the electron transport chain, which takes electrons from NADH to pass on to coenzyme Q. Iron-sulfur clusters are common cofactors. These metal coenzymes are held in place by four cysteines or by two cysteines and two histidines. Because the cysteines are forming covalent bonds with the irons in the iron-sulfur cluster, the clusters are classified as cofactors. An example of this is in ferrodoxins. The iron-sulfur cluster aids in the transfer of electrons.
Zwitterion
A dipolar version of an amino acid wherein positively and negatively charged functional groups cancel one another out, resulting in a neutral ion.
Lineweaver-Burk Plots
A double-inverse graph of the reaction rate (v inverted to 1/v) and substrate concentration ([S] inverted to 1/[S]) graph described above. Y-intercept = 1/Vmax X-intercept = -1/Km
Michaelis-Menten Saturation Curve
A graph of reaction velocity vs. substrate concentration [S]. This graph reveals the relationship between 1/2 Vmax and Km, as well as the overall concept of "saturation kinetics".
Solvation layer
A layer of water that surrounds a dissolved protein. The water molecules in this layer interact closely with each other and with the protein's surface. The water in the hydration layer is more ordered that the bulk water in the general area and is considered not to participate with the bulk (a.k.a, unstructured) water when considering colligative properties.
Provide a conceptual explanation for: substrate, active site, and enzyme-substrate complex.
A substrate is a molecule that is acted upon by an enzyme. A substrate could be a small molecule, a protein, a lipid, DNA, etc. The key here is that a substrate is a molecule that is converted to a product by an enzyme. An active site is the part of an enzyme that where the substrate is converted to product. It is typically a small port or pocket that will hold only that enzyme's very specific substrate. There are often cofactors associated with the active site, such as metal ions or small molecules that assist in the catalysis of the enzyme. The amino acids that form the active site have specific properties associated with both the type of substrate and the type of reaction being catalyzed (as discussed in questions 8 and 9). The active site likely changes conformation as the substrate binds, as described by the induced fit model of enzyme kinetics. The enzyme-substrate complex is formed when the substrate is bound in the active site. Once the enzyme- substrate complex is formed, the substrate will be converted to product (going through the transition state first). This is represented by the basic kinetics formula E + S ↔ ES → EP ↔ E + P
Protein Folding
A translated protein assumes secondary structure almost instantly, and then folds into its globular or structural tertiary state, driven by the interactions described below. You may see proteins in various states of folding referred to as: Globule (fully folded) Molten Globule (partially folded) Molten (fully unfolded; a.k.a denatured)
Draw isoleucine and glutamic acid in their zwitterion forms.
A zwitterion has both positive and negative charge. Free amino acids will exist as zwitterions at neutral pH because both the amine group and the carboxyl group will be deprotonated (giving the amine group a positive charge and the carboxyl group a negative charge). The zwitterion itself is neutral.
A student discovers a mutation in the binding pocket of an enzyme which changes an alanine to a lysine. The mutant enzyme is much more efficient than the wild type. This suggests that the substrate is:
A. Positively charged B. Negatively charged C. Polar D. Non-polar
Which amino acid would help stabilize the binding pocket for an enzyme that catalyzes the conversion of ethanol to acatealdehyde?
A. Valine B. Proline C. Tyrosine D. Lysine
Protein Denaturing Agents
Acid Heat Urea Mercaptoethanol
"Amino acid characteristics" Absolute Configuration:
All amino acids are designated as either L- or D-, depending on the side on which the amino group is located in a fischer projection (L = left; D= Right). All native human animo acids are L-amino acids. L- and D- do NOT correlate directly with R and S and should be considered as separate stereochemical designations. Most L-amino acids are S, but some L-amino acids are R (ex. cysteine).
-R groups DETERMINE chemistry
Amino acid -R groups largely DETERMINE the chemistry of the amino acid, and the combination of -R groups in a protein almost exclusively DETERMINES its chemistry and folding pattern.
Quaternary Protein Structure
Association of multiple folded proteins into a multi-subunit complex. Hemoglobin = classic example of quaternary structure. Consists of four protein chains, two alpha subunits and two beta subunits. Each subunit contains one heme capable of binding one O2 molecule.
Describe the difference between a catalyst and an enzyme.
Both catalysts and enzymes increase the rate of a reaction by lowering the activation energy. Enzymes, however, are organic molecules, while catalysts can be inorganic molecules. Neither catalysts nor enzymes are consumed during a reaction; both can be recycled and used again. Enzymes are highly specific for a specific substrate or group of substrates, while catalysts can be more universal. Catalysts are often just metal ions or other small molecules that activate the substrate. Reactions using enzymes are millions of times faster than without, while reactions with catalysts are typically not increased at that speed. ALL enzymes are catalysts, but NOT all catalysts are enzymes.
Draw both resonance structures for a peptide bond.
Both resonance structures for the peptide bond are shown. Notice that they are not equally favored, so one will be a more significant contributor to the hybrid than the other.
Proline turns
Can be considered as either disrupting 2 structure or as contributing to 3 structure. Neither a-helices, nor b-sheets can contain proline internally without disruption of the 2 structure. However, proline residues are often found at the beginning of a-helices and are very common (along with glycine) in the sharp turns at the end of two adjacent rows in a b-sheet.
Identify one real-life (preferably human-body) example of competitive inhibition.
Competitive inhibition - the inhibitor resembles the substrate and fits into the active site reversibly, competing for active site occupancy with the substrate. An example of competitive inhibition is statin drugs. These drugs are HMG-CoA reductase inhibitors used to lower cholesterol levels by inhibiting the enzyme's function of producing cholesterol in the liver. The statin drugs inhibit HMG-CoA reductase by occupying the enzyme's active site, prohibiting HMG-CoA to enter and preventing the enzyme from converting HMG-CoA to mevalonic acid, which is an irreversible step in the cholesterol synthesis pathway. Because statins bind the active site reversibly, they are competitive inhibitors and not irreversible inhibitors.
WHEN YOU SEE ENZYME THINK
Enzyme = Protein
ENZYME MNEMONIC
Enzymes help reactions Over The HILL (energy of activation)
How do enzymes affect each of the following ? a) reaction rate b) energy of activation c) equilibrium d) Keq e) yield f) percent yield
Enzymes increase reaction rate, lower activation energy, and do NOT affect equilibrium, Keq, yield, or percent yield. However, without an enzyme, it could take years, even centuries or millennia, for a reaction to reach equilibrium and thus reach the yield that could be achieved in seconds with the enzyme present.
Important Note
Enzymes increase reaction rate, lower the energy of activation, catalyze both the forward and reverse reactions, are NOT consumed in the process, and NEVER alter the thermodynamic properties of the reaction. The most notable difference is that, as proteins, enzymes are far more sensitive than inorganic catalysts to environmental conditions such has temperature and pH.
Entropy and protein folding
Even when water interacts with a dissolved polar solute, this interaction is less entropically favorable than those same water molecules interacting with only other water molecules. However, the driving thermodynamic force that favors protein folding results from the fact that non-polar regions require a much GREATER ordering of water molecules to accomplish solvation. Therefore, transitioning from solvation of non-polar regions to solvation of a mostly polar or charged globular protein surface, represents a net increase in entropy. In fact, it is enough to overcome the decreased entropy associated with the protein being in a folded rather than an unfolded state. This favorable increase in entropy is a major contributor to the overall conformational stability of the folded protein.
Vitamins
Fat soluble = A, D, E and K Water soluble = all the rest
Salt bridges
Formed when acidic and basic -R groups undergo neutralization reaction resulting in a salt.
Tertiary Protein Structure
Geometric, three-dimensional folding of the alpha helices, beta sheets, and other moieties to form a functional globular or structural protein.
Draw a titration curve for the titration of a solution of phenylalanine with sodium hydroxide. Describe the relative concentrations of each species at each of the above-stated points along the curve. Draw a similar titration curve for aspartic acid titrated with sodium hydroxide. In what ways does this titrations curve differ?
Half-equivalence point and buffer region: concentration of NaOH is much lower than phenylalanine Equivalence point and pI: concentration of NaOH is equal to phenylalanine End point: concentration of NaOH is much higher than phenylalanine. Aspartic acid has a double curve because the side chain is charged. Because of this, as the pH rises due to the OH- in solution (from the dissociation of NaOH), two different equivalence points are reached, as the two acid groups are deprotonated. The two points are at different pH's because they have different pKas.
Hydrogen bonds
Hydrogen bonding between -R groups also encourages folding and stabilizes the folded protein.
Secondary protein structure Beta sheets:
Hydrogen bonding between ALL of the carbonyl oxygens in one row and the amide hydrogens in the adjacent row. ALL residues involved in hydrogen bonding!!!! R groups are directed perpendicular to the plane of the beta sheet, on both sides. Beta sheets assume a PLEATED conformation. This is necessary for the carboxyl and amide moieties to line up properly so that every residue is participating in two hydrogen bonds.
How will a protein fold?
Hydrophobic -R groups fold INTO the protein core (hydrophobic environment), and hydrophilic -R groups are more common on the surface of the protein (hydrophilic environment).
"Protein Folding" Hydrophobic Core
Hydrophobic -R groups fold into the interior of a globular protein to escape water. They often bring some smaller polar groups with them, which interact in a complementary way to stabilize the folded protein further.
Which amino acids are most likely to be part of an enzyme active site where the first step of the catalyzed reaction involves abstraction of a proton from the substrate? What if the first step is protonation of a functional group on the substrate?
If the first step of the reaction involves extraction of a proton, one can predict that two amino acid residue types are likely in the active site: a) at least one amino acid that performs the enzymatic function of abstracting the proton, and b) amino acids that stabilize the product of that deprotonation. In the first case, it is most likely that an amino acid is involved that features a basic side chain. This would be histidine, arginine or lysine. In fact, histidine in particular, is frequently a part of enzyme active sites for this very reason. In the second case, the active site amino acids will need to hydrogen bond with the atom losing the proton. The amino acids will be donating the hydrogen to the hydrogen bond (since the substrate is losing its proton). Likely amino acids are lysine and arginine, because the amine groups can readily hydrogen bond with the substrate which is now likely negatively charged (due to losing a proton). By contrast, if the first step were protonation we would be looking for amino acids with acidic side chains, such as glutamate or aspartate.
Non-competitive Inhibition
Inhibitor binds AWAY FROM the active site and changes the shape of the enzyme. The inhibitor has an equal affinity for both the enzyme-substrate complex (E-S) and the enzyme (E). (Substrate attached to active site of enzyme and inhibitor at another site of the enzyme). Vmax = DECREASES Km= NO CHANGE
Uncompetitive Inhibition
Inhibitor binds ONLY with the enzyme-substate complex. Vmax= DECREASES Km= DECREASES
Competitive Inhibition
Inhibitor binds at the active site; the inhibitor resembles the substrate in shape; the inhibitory effect can be overcome by increasing the concentration of the substrate. Vmax= NO CHANGE Km= INCREASES
Irreversible inhibition
Inhibitor binds covalently to the enzyme and/or the active site, disabling the enzyme for either a prolonged period of time, or permanently.
Mixed
Inhibitor has unequal affinity for the E-S and the E, favoring one over the other. Vmax= DECREASES Km= DECREASES if inhibitor = increased affinity for E-S over E Km= INCREASES if inhibitor = increased affinity for E over E-S
Reversible Inhibition
Inhibitor is not permanently bound; enzyme is not completely disabled. Four types: competitive, uncompetitive, non-competitive, and mixed.
Electrostatic interactions
Interactions between charged -R groups both encourage the act of folding itself, and stabilize the protein in its folded state.
Protein separation techniques
Isoelectric point Electrophoresis
Can a person more easily overdose on fat-soluble vitamins or water-soluble vitamins? Why?
It is much easier to overdose on fat soluble vitamins than water soluble vitamins. Ingesting large amounts of water soluble vitamins is generally not harmful because they will quickly be dissolved and excreted through the urine. Fat soluble vitamins, however, will be stored in fat tissue and membranes. Ingesting large amounts of these vitamins can lead to an overdose because disposal of the excess is more difficult.
What steps are necessary to cause a simple denatured protein to re-fold? Will this process work, unaided, for all proteins?
It is often not possible to re-fold unfolded proteins. In cases where you can, proteins have been slowly denatured using a mild denaturant, something that will break hydrogen bonds and disulfide bonds but not other covalent bonds. When denaturing a protein that you want to re-fold, you want to denature slowly. Too quickly or too harshly and irreversible denaturing will occur, such as collapse of all hydrophobic amino acids into the center of the protein. To refold a denatured protein, you want to slowly remove the denaturant from solution. As you slowly wash away the denaturant, secondary structure will start to return, followed by tertiary and quaternary. This will not work with proteins that have been heat denatured, as this often disrupts hydrophobic interactions to the point that all hydrophobic residues collapse together. This will create hydrophobic interactions so strong that it cannot be unfolded. Other types of denaturation that is difficult or impossible to reverse: proteins that are subject to extremes in pH or harsh denaturants that may dissolve covalent bonds.If the first step of the reaction involves protonating the substrate, the active site amino acids will need to hydrogen bond with the atom gaining the proton. In this case, the amino acids will be accepting the hydrogen in the hydrogen bond (since the substrate is gaining its proton). Likely amino acids are aspartic acid and glutamic acid, since the carboxylic acid groups can readily bond with the new proton.
Common applications of secondary structures
Keratin, found in hair and nails = alpha helices. Fibroin, the molecule that makes up silk = beta sheets.
Positive Cooperativity
Ligand affinity increases with the binding of each subsequent ligand. In the case of hemoglobin, affinity for the first oxygen is relatively low, but increases for the second, third, and fourth oxygen to bind. Therefore, the first oxygen (highest cooperative affinity) dissociates at the slowest rate, but each subsequent oxygen is released more easily.
List all amino acids that could be logical "stabilizing features" of the enzyme pocket for an enzyme whose substrate is: a) positively charged, b) negatively charged, c) polar, or d) non-polar
MCAT-2015 has already demonstrated a penchant for this question type. They will describe a feature of an enzyme active pocket, or a substrate, and ask you to predict the amino acid involved. You are only looking for COMPLENTARITY. Generally speaking, opposite charges will attract and stabilize each other, polar molecules will be attracted to each other and stabilize one another, non-polar moieties will aggregate away from water stabilizing the molecules involves, and any functional group capable of hydrogen bonding with another functional group will form a strong stabilizing association. For H-bond associations remember to consider whether the functional group can act as an H-bond donor, and H-bond recipient, or both. More specifically, for this question, think about the types of interactions that will stabilize something charged, polar, or non-polar. A. Positively charged substrates = aspartic acid, glutamate, asparagine, glutamine - charge interactions +/-, hydrogen bonding with carboxyl group of asparagine and glutamine. B. Negatively charged substrates = lysine, arginine, serine, threonine, tyrosine, asparagine - charge interactions +/-, hydrogen bonding with the alcohol group of ser, and thr, hydrogen bonding with amine group of tyr, asn C. Polar substrates = asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine - hydrogen bonding with amines and acid groups D. Non-polar substrates = alanine, glycine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan - hydrophobic interactions.
Peptide chain conventions
Peptides are written, read AND synthesized from N-terminus --> C-terminus
What is double bond character?
RIGID peptide bond with LIMITED rotation.
Michaelis Constant: (Km)
Relative measure of an enzyme's affinity for its substrate. Km= [S] @ 1/2 Vmax
Resonance
Resonance between the pi electrons of the C=O bond, and the nitrogen lone pair of the C-N bond, yield two resonance structures for any peptide bond. The actual structure is a hybrid of the two, and therefore: BOTH the C=O bond and the C-N bond in a peptide bond have DOUBLE BOND character.
Explain conceptually, the process of separating proteins via electrophoresis.
Separating proteins by electrophoresis will separate proteins based on size. To perform electrophoresis, proteins are placed in a solution with a detergent to denature them and to coat them uniformly with a negative charge. This gives proteins a uniform charge to mass ratio. Proteins are then run through a gel of polyacrylamide, which will slow down the migration of larger proteins more than smaller ones. The gel is run from NEGATIVE to POSITIVE electrodes, so the proteins are pulled toward the positive pole due to the negative charge from the detergent. The SMALLEST proteins will run FURTHEST into the gels, while the largest proteins will stay near the top.
Explain conceptually, the process of separating proteins via isoelectric point.
Separating proteins by isoelectric point is called isoelectric focusing. A gel is created with stable pH gradient. A protein in a region of the gel with a pH lower than its isoelectric point will be positively charged (because it will be fully protonated) and so will move toward the negative cathode. A protein in a region with a pH higher than its isoelectric point will be negatively charged (because it will be fully unprotonated) and so will move toward the positive anode. As the protein moves through increasing pH in the gel, the protein's charge will decrease until it reaches the pH of its pI, at which point it will become neutral. At this point the protein will cease to move through the gel, because it has no charge and so has no pull toward either electrode. This causes proteins to form very sharp bands at the pH equal to each protein's pI.
Name six molecular interactions that contribute to the 3 protein structure.
Six interactions between amino acids that contribute to tertiary protein structure are: 1 = hydrogen bonding - non-covalent bond between either backbone atoms (N-H or C=O) or side chains (amine groups, carboxyl groups, alcohol groups) 2 = disulfide bonds - covalent bond between the sulfurs of two cysteine residues 3 = hydrophobic/hydrophilic interactions - in soluble proteins, the hydrophobic amino acids will collapse into the protein core. In membrane proteins, the hydrophilic membranes will be either outside the membrane in the cytoplasm or inside the core of the protein, away from the membrane bilayer, with hydrophobic amino acids located within the membrane bilayer. 4 = ionic interactions (salt bridges) - charge-charge interactions between a positively charged amino acid and a negatively charged amino acid 5 = Van der Walls forces - intermolecular forces that repel atoms away from each other (steric hindrance) 6 = Proline turns - because of proline's unusual cyclical shape, introducing a proline into an alpha helix or beta sheet will cause a kink. It can also aid in beta turns.
Enzyme classification by reaction type
Some enzymes fit into multiple categories. Oxidoreductases = REDOX reactions Transferases = Transfer of a functional group (ex. kinases, aminotransferases). Hydrolases= hydrolysis Isomerases = rearrangements (ex. phosphoglucose isomerase [G6P -> F6P], epimerases) Lyases = AB <-> A + B [cleavage/synthesis; NO H2O, NOT hydrolysis] Ligases = addition or synthesis of LARGE molecules, usually ATP-dependent (ex. DNA ligase)
"Peptide Bond Formation" Reaction type: Dehydration synthesis and acyl substitution.
The amine group nitrogen (nucleophile) from the NEW amino acid attacks the carbonyl carbon (electrophile) on the C-terminus of the growing peptide chain (aided by the enzymatic function of the ribosome).
Provide a conceptual definition for isoelectric point. To which acid-base titration term is the isoelectric point most similar? a) half- equivalence point, b) equivalence point, c) end point, or d) buffer region? Why?
The isoelectric point (pI) is the pH at which a molecule carries no net charge. For amino acids and other organic molecules, the molecule is often in the form of a zwitterion. For molecules that have two pKas, the equation for pI is: pI = (pKa1 + pKa2)/2 At pH lower than the pI, the molecule will have a net positive charge. At a pH higher than the pI, the molecule will have a net negative charge. This can be utilized to separate molecules based on their pIs by varying the pH in a gel. The isoelectric point is most similar to the equivalence point in acid-base titration. In acid-base titration, the equivalence point is the point at which all of the starting solute is neutralized by the titrant. At that point the acid and the base are present in equal quantities. This is similar to the isoelectric point, because in both cases the acid and the base are both neutralized at that specific pH, rendering a net charge of 0.
Hydrophilic surface
The majority of the -R groups on the surface of a globular protein are either polar or charged.
What aspect of a protein is primarily responsible for the manner in which it folds?
The primary sequence of a protein is primarily responsible for how it folds. There is evidence for this in the fact that simple proteins fold spontaneously without chaperone proteins. Also, as peptides are exiting ribosomes, they immediately begin folding. This makes sense if you think about it: certain amino acids are found in certain secondary structures, and some groups of amino acids found together will almost always fold in the same way. Mutations in key amino acids can completely destroy secondary and tertiary structure, while other mutations may alter only segments of folding.
Order of Deprotonation
There are multiple cases where you will benefit from knowing approximate pKa values for the side chains. There are not that many, and they predict the order in which the acidic functional groups of a protein or amino acid will be deprotonated.
While true that L- and D- do not correlate directly with R and S, among all 20 of the common amino acids there are only two cases in which an amino acid cannot be said to be BOTH L- and S. Name the two exceptions and explain why, specifically, they are exceptions.
To answer this question, first we need to define what L, D, R, and S are. For amino acids, L and D refer to the glyceraldehyde molecule that the amino acid could theoretically be synthesized from (D-glyceraldehyde or L- glyceraldehyde). R and S refer to the absolute stereochemistry of the molecule. To designate a molecule as R or S, you must rank each R group of a chiral carbon for priority based on atomic number of the atom connected to the carbon. The lowest priority R group is pointed away from you. You then consider the priority of the other three R groups. If that priority descends in a clockwise fashion, the molecule is R. If it descends counterclockwise, then the chiral center is S. For almost all the amino acids, the L designation and the S designation occur together. This makes sense, because if they all could theoretically derive from the same glyceraldehyde molecule, they would all end up with the same stereochemical orientation. Two amino acids, however, differ from this rule. One is glycine. The tetrahedral carbon of glycine is not a chiral center, because it has two hydrogens attached and so does not have four different R groups. Because it does not have a chiral center, glycine cannot be designated as either R or S. Cysteine is the other oddity. Because cysteine has a sulfur at the second position in its side chain, the side chain has a higher ranking than other side chains when considering whether to designate it as R or S (due to cysteine's higher atomic mass). This means that L - cysteine will be R - cysteine, because the sulfur has changed which direction the priorities of the R groups turn.
Protein Hydrolysis
Trypsin and chymotrypsin cleave proteins on the CARBOXYL side of specific amino acid residues: Trypsin: arginine, lysine Chymotrypsin: phenylalanine, tryptophan, tyrosine
Disulfide bonds
Two oxidized CYSTEINE residues form a disulfide (R-S-S-R) bond. This is the strongest type of protein folding interaction. Disulfide bonds between keratin alpha helices are what make hair more or less curly.
"Amino acid characteristics" Alpha-carbon stereocenter:
all human amino acids, except one (glycine), are chiral at the alpha carbon because the alpha carbon contains four different substituents: an -R group, a hydrogen, a carboxylic acid, and an amine.
Proteins
amino acid polymers; amino acids are often called residues; oligopeptide = very small chain of amino acids; polypeptide = longer chain of amino acids.
Two theories of enzyme specificity are:_______and _____&______. Which of these two theories has been largely dismissed by scientists? Why has it been dismissed?
Two theories of enzyme specificity are: induced fit and lock and key. The induced fit model predicts that the active site's empty structure is not an exact fit for the substrate, and may be rather nondescript. As the substrate begins binding the pocket, small but specific conformational changes occur, such that until the substrate is completely bound, the final shape and charge characteristics of the active site are not in place. This is the FAVORED theory: induced fit means that as the substrate binds, the affinity for the substrate increases. The resulting conformational changes will induce a higher affinity for the transition state, stabilizing it and lowering activation energy. The lock and key model predicts that the active site of an empty enzyme is an exact fit for its substrate; the substrate is the key, and the enzyme active site is the lock. This model is NOT FAVORED by scientists because it predicts a very rigid, inflexible active site. Energetically, this would be unfavorable, and sterically for many enzymes this would be unlikely or impossible, based on where the active site is located. While this model is unlikely to be correct for most enzymes, there are a few for which the energy profiles of binding, as well as their 3D structures, indicate that the active site is more rigid than most enzymes.
Identify one real-life (preferably human-body) example of uncompetitive inhibition.
Uncompetitive inhibition - the inhibitor binds the enzyme only after the enzyme-substrate complex is formed and does not bind the active site. Uncompetitive inhibition is incredibly uncommon, possibly because of the way this inhibition works. If the inhibitor can bind only after the enzyme-substrate complex forms (but must act before substrate is converted to product), then depending on the rate of the enzyme, the time window for inhibitor action is very limited. Lithium, a drug used to treat manic depression, has been shown to act as an uncompetitive inhibitor in the phosphoinositide synthesis pathway, inhibiting inositol monophosphatase and thus preventing inositol recycling in the brain.
Proline
Usually the first residue at the very end of an alpha helix, but rarely found inside the helix because it introduces a KINK/TURN. This same KINK/TURN is desirable at the end of beta-sheets because the chain must make a 180 degree turn to align as a neighboring row in the beta sheet.
What are the differences between vitamins and minerals? What key biological functions involve or require vitamins and minerals?
Vitamins are relatively small, organic molecules that are essential nutrients required in small amounts for proper metabolism. Many cofactors and coenzymes are derived from vitamins, such as NAD discussed in the previous question (derived from the vitamin niacin). Humans have lost the ability to synthesize vitamins in sufficient quantities in our bodies, and so we must acquire them through our diet. Water soluble vitamins are the ones most often required for synthesis of cofactors. Fat soluble vitamins are needed for diverse biological functions, such as vitamin A, which is converted to retinal and used as a coenzyme in visual proteins. Other fat soluble vitamins act as antioxidants, aid in maintaining blood pressure, and play a role in blood clotting. Minerals are inorganic elements or compounds necessary for bone formation (calcium and phosphate), ion gradients (sodium and potassium), oxygen transport (iron-containing heme), muscle contraction (calcium), ATP processing (magnesium), production of stomach acid (chlorine), etc. Minerals are gained through diet and are needed in very small quantities, making them micronutrients.
Michaelis-Menten Saturation Curve (graph explained)
Vmax is the rate of the reaction at saturation levels of substrate. This is the theoretical maximum rate of the reaction, although in practice the reaction will never reach this velocity. At low substrate concentrations, the rate of the reaction increases quickly. As more and more substrate is added, the rate increase slows down. This is because at low substrate concentrations, there is a much higher chance of free substrate encountering an empty enzyme active site, but as substrate concentration increases, that likelihood continuously decreases. At max saturation, the velocity will hold steady, as the enzyme molecules in the reaction are always occupied with a substrate molecule (as soon as a substrate molecule is converted to product, a new substrate molecule is picked up). Imagine a room with 50 chocolate vendors milling around in it (enzymes) that will exchange one chocolate bar for a dollar. Buyers (substrates) are slowly allowed to enter the room. At the beginning, it is easy for a buyer to bump into an unoccupied vendor, because there are fewer buyers than vendors. As more and more people enter, though, the room gets crowded, and eventually every vendor is occupied with a buyer. The rate at which the vendors can exchange a chocolate bar for a dollar at this point is Vmax because the vendors have reached a saturating concentration of buyers. Km is the Michaelis constant, the substrate concentration at half Vmax. This constant is used in calculations in Michaelis-Menton kinetics, to calculate the rate of a reaction at a given concentration of substrate using the equation
When you see PROTEIN or ENZYME think:
What amino acids are present and what is the chemistry of their -R groups?
Important Note
With the exception of amino acids that have charged -R groups (Asp, Glu, Lys, Arg, His), ALL of the amino acids exist as zwitterions at a pH of 7.4. This can be very confusing because textbooks NEVER draw them this way. Most texts draw them in their "non-ionized" form, whith -COOH and -NH2 groups. That combination DOES NOT EXIST at physiological pH, or at ANY pH!!! Below a pH of about 9 the amine group will be protonated: -NH3+. Above a pH of 9 the amine group will be -NH2 (as shown in most texts), but at that very high pH the caboxyl group will have long ago been deprotonated: -COO- (pH =2).
NON-ESSENTIAL Amino Acids
Your body can synthesize this amino acid on its own.
ESSENTIAL Amino Acids
Your body cannot synthesize this amino acid. You must ingest it.
Cofactors
a general term for any species required by an enzyme to function; coenzymes and prosthetic groups are both examples of cofactors.
How do each of the following affect reaction rate for an enzyme-catalyzed reaction? a) pH b) temperature c) substrate concentration d) enzyme concentration
a) All enzymes have an optimal pH at which enzyme activity is highest. For most enzymes this is around 7, but the optimum pH can vary based on typical pH for that enzyme's environment. Pepsin, for example, has a pH optimum of about 2 (the normal pH of the stomach). Enzymes in lysosomes will also prefer a lower pH, as will the enzymes of acidophiles. Whatever the optimum is, movement in either direction causes a rapid decrease in rate because changes in pH will affect the hydrogen bonding of the enzyme itself, possibly altering the structure, and will likely disrupt the enzyme-substrate complex. b) Mildly increasing temperature will increase the rate of an enzyme catalyzed reaction. However, increasing the temperature too much will denature the enzyme, causing the reaction rate to drop precipitously. c) At low substrate concentrations, the reaction rate will increase rapidly. As more and more substrate is added, the rate increase will drop off, as described in question 23. d) Enzyme concentration shows a "saturation curve" similar to that for substrate concentration and similar logic applies. Adding enzyme when enzyme concentration is low will increase the rate, because there is plenty of substrate available for the newly added enzyme molecules to act upon. As saturation levels are reached, the rate increase will drop off because enzymes are less and less likely to encounter a substrate molecule.
Estimating Isoelectric Point
acidic, basic and neutral amino acids. pI neutral = average of pKa -amine group and pKa -carboxyl group. pI acidic = average of pKa -acidic R group and pKa-carboxyl group. pI basic = average of pKa -amine group and pKa - basic R group.
structural proteins
actin (thin filaments, microfilaments), tubulin (microtubules), keratin (hair and nails, intermediate filaments), elastin (connective tissue, extracellular matrix).
Mechanism of catalysis
cofactors, coenzymes and prosthetic groups.
Binding proteins
hemoglobin, calmodulin, troponin, tropomyosin, histones, transcription factors, cell adhesion molecules.
Secondary protein structure Alpha-Helices:
hydrogen bonding between the carbonyl oxygens and the amide hydrogens that are exactly FOUR residues apart. ONLY every FOURTH residue is involved in Hydrogen bonding. R groups are directed exactly away from the alpha helix cylinder (ex. perpendicular to a plane tangent to the surface of the alpha helix).
Motors
myosin (power stroke, cellular transport), kinesins and dyneins (vesicles, cellular transport, cell division, cilia, flagella).
Primary protein structure
the amino acid sequence.