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105 Cards in this Set

  • Front
  • Back
Define/explain the following term:

Active Site
It is a relatively small part of the whole enzyme structure.

It is a 3-Dimensional cleft with participating components from different parts of the primary structure.

It's binding uses multiple weak interactions including:

Hydrogen bonds, Salt links, Van der Waals interactions, hydrophobic effect
Define/explain the following term:

Enzyme-Substrate Complex
It is what is created when enzymes form complexes with their substrates (binding). It is a unique microenvironment for the reaction to proceed.
Define/explain the following term:

Induced Fit
It pertains to the conformational changes giving tighter binding in a new conformation.
Define/explain the following term:

Initial Velocity
It is the velocity measured at the beginning of a reaction.
Define/explain the following term:

Steady State
Steady state exists when the concentration of the [ES] complex is constant.

The rate of formation of ES equals the rate of breakdown of ES (forward + backward). This is the steady state assumption.
Define/explain the following term:

Vmax is defined as the maximum velocity of a reaction.
Define/explain the following term:

It is the substrate concentration at which velocity is exactly 1/2 of maximum velocity (Vmax).
Define/explain the following term:

It is the catalytic efficiency. It has units of sec-1.
Define/explain the following term:

Turnover Number
It is the number of substrate molecules converted into product by one enzyme active site per unit time, when the enzyme is fully saturated with substrate.
Define/explain the following term:

It is the equilibrium dissociation constant for the [ES] complex.
Write out a simple Michaelis-Menten kinetic mechanism for an enzyme-catalyzed reaction.
The Michaelis-Menten Equation
Recognize the Michaelis-Menten equation, and sketch a graph of Vo vs. [S] for an enzyme-catalyzed reaction that illustrates Vmax and KM.
Vo vs. [S]
Explain the definition of KM in terms of the rate constants in the Michaelis-Menten kinetic mechanism, but then give the operational definition of KM that holds no matter what the actual kinetic mechanism is for a particular enzyme.
It is the substrate concentration at which velocity is exactly 1/2 of the maximum velocity.

It is an aggregate constant, meaning it is the sum of rate constants for breakdown of ES divided by rate constant for formation of ES.

If k2 << k-1, then it equals k-1/k1 = Kes, and it can then be taken as a measure of the dissociation constant for the ES complex.
Explain the relationship of Kcat to Vmax.
Relationship between Kcat, Vmax, and [ET].
Explain the relationship of Km to Kes.
Km is an aggregate rate constant for breakdown and formation of a complex, while Kes is just the rate constant for dissociation.
What are the units of Km?
It has units of M.
What are the units of Kcat?
It has units of inverse time, sec-1 or min-1.
What are the units of Vmax?
It has units of velocity, concentration/time (conc*time-1).

E.g. mM/min
Express the ratio of occupied active sites to total enzyme active sites ([ES]/[ET]) in terms of Vo and Vmax.
Ratio of Occupied Sites / Total Active Sites
What is the maximum possible value of the ratio between occupied active sites and total enzyme sites?
Given a plot of Vo/Vmax vs. [S], find the value of Km from the plot.
Since Km is the substrate concentration at which the velocity (V) is exactly 1/2 of the maximum velocity (Vmax), it can be derived by looking at the graph and finding the point on the curve that the velocity is 1/2 the Vmax. Draw a vertical line to the base of the X-axis, and you have Km.
What two things is the parameter Kcat/Km used to indicate?
It is a measure of an enzyme's substrate preference and enzyme's catalytic efficiency.
What sets the upper limit for the value of
Kcat/Km for an enzyme?
k1 sets an upper limit on the value for it.
What is the approximate range of values for that upper limit of Kcat/Km? (Give the units of those values, too.)
The range can be very large or very small, dependent upon the rate of k1. If k1 is large, Kcat/Km could be large as well, but if k1 is small, Kcat/Km is limited.

Specifically, maximum (diffusion-limited) k1 = ~ 10⁸ - 10⁹ M⁻₁ * sec⁻₁
Define cooperative substrate binding.
It is the binding of substrate to 1 active site affecting the properties of other binding sites (on other subunits) of the same enzyme molecule.
Define sigmoid plot.
It pertains specifically to a Vo vs. [S] curve. It is used as a diagnostic of cooperativity.

Velocity increases steeply in [S] range around the apparent "Km" ([S] where velocity = 1/2 Vmax), so in that substrate concentration range (around Km), a small change in [S] makes a big change in velocity.

Regulatory consequences: binding another molecule (an allosteric inhibitor or activator).
What shifts the sigmoid plot to the right?
An allosteric inhibitor will shift the plot to the right.

The velocity is less at a given [S], requires more [S] to reach 1/2 Vmax.
What shifts the sigmoid plot to the left?
An allosteric activator shifts the plot to the left.

Velocity is greater at a given [S], requires less [S] to reach 1/2 Vmax.
What does the Michaelis-Menten equation actually measure?
It measures the dependence of velocity on [substrate] for many enzymes.
What are the three kinetic parameters?
Km, the Michaelis Constant;
Kcat, the turnover number;
Kcat/Km, the catalytic efficiency of an enzyme.
What is binding known as?
The essence of enzyme action!
What are the multiple weak interactions that binding uses?
Hydrogen bonds,
van der Waals,
hydrophobic interactions,
and salt links.
What does the specificity of binding depend on?
It depends on active site crevice being sterically and chemically precisely complementary to the groups it is binding.
What is the solution to the problem inherent in measuring reaction rates of substrates and products?
Measure the velocity at early times in the reaction, before [S] decreases significantly (Do this when [P] ~ 0).
What type of graph does a simple uncatalyzed S --> P reaction provide?
It yields a straight line plot of Vo vs [S].
At low [S], how does the initial velocity relate to [S]?
Vo is proportional to [S] at low [S].

If you double [S], it leads to a double in Vo.
What does Kcat equal in the Michaelis-Menten kinetic mechanism?
Kcat = K2.
Describe cooperativity.
Binding of substrate to 1 active site affects the properties of other binding sites (on other subunits) of the same enzyme molecule cooperative substrate binding
Describe sigmoid plot.
It starts slowly, then increases quickly and becomes almost vertical, and then it levels off
Describe allosteric enzyme
They are multisubunit enzymes (with more than one catalytic subunit, so >1 active site/enzyme molecule)
Describe double
reciprocal plot
A linear line representing 1/V vs. 1/[S].
Describe reversible inhibition
competitive or noncompetitive, reversible
Describe irreversible inhibition
Inhibition that is not reversible. Uses one of the following means:

1) group-specific covalent modifying agents
2) suicide inhibitors (mechanism-based inhibitors)
3) affinity labels
4) transition state analogs
Describe affinity label
Structural similarity to substrate "guides" reagent to active site.
The reaction at active site covalently inactivates enzyme
Describe suicide substrate
Exhibits structural similarity to substrate "guides" reagent to active site.
The enzyme TREATS IT AS A SUBSTRATE, starting chemical catalytic process with the inhibitor.
chemical mechanism itself leads enzyme to react covalently with inhibitor, thus "committing suicide"
Describe mechanism-based inhibitor
Depends on chemical mechanism of catalysis.

Example: penicillin (inhibits an enzyme, a transpeptidase, required for bacterial cell wall synthesis)
Describe transition state analog
structurally similar to transition state, which binds even more tightly to enzyme than substrate binds, so very high affinity for active site.

Useful for:

* understanding catalytic mechanisms (clues about structure of transition state)
* very specific inhibitors of enzymes (pharmaceutical applications)
* antigens for immunizing lab animals to generate antibodies with binding sites complementary to the transition state such that the antibodies themselves have catalytic activity ("abzymes")
How do you calculate the Vmax and Km from a Lineweaver-Burk plot?
Y-intercept = 1/Vmax
Slope = Km/Vmax
X-intercept = 1/Km
Explain competitive and noncompetitive inhibition in terms of a diagram of linked reaction equilibria for formation of ES, EI, and (if it can form) EIS.
In competitive inhibition, the hyperbolic graph will decrease as you add more inhibitor. Because it is competitive however, you will eventually be able to overcome the inhibitor by adding increasingly large amounts of substrate. This is not the case for noncompetitive inhibition, wherein a high [S] does not overcome the effect of the inhibitor.
Explain how these two types of inhibition can be distinguished from each other graphically, on a Vo vs. [S] plot
To distinguish between the two on a Vo vs. [S] plot, you must see if the Vmax or Km is altered. If the Vmax does not change and Km does, it is competitive inhibition. If Km does not change and Vmax does, it is noncompetitive inhibition.
Explain how these two types of inhibition can be distinguished from each other graphically, on a double reciprocal (Lineweaver-Burk) plot.
In a double recriprocal plot, you must check the X and Y intercepts. If the Y intercept does not change but the X intercept does, you are looking at competitive inhibition. If the Y-intercept changes but the X-intercept doesnt, you are looking at noncompetitive inhibition.
Why is ethanol used as an antidote for ethylene glycol (antifreeze) poisoning?
Toxic effects of ethylene glycol and of methanol depend on their -OH groups being oxidized to aldehyde (by alcohol dehydrogenase in body) and then to carboxylic acids
Ethanol (another substrate with less toxic oxidation products) competes for binding to alcohol dehydrogenase
If alcohol dehydrogenase molecules are all occupied with ethanol as a substrate, ethylene glycol (or methanol) passes through body without being oxidized and is excreted (kidneys)
What type of inhibitor is pencillin?
It is an example of an inhibitor that is both a transition-state analog and a suicide substrate.
Give some other specific examples of reversible and irreversible enzyme inhibition.
Ibuprofen (Motrin, Advil) is a reversible inhibitor
Aspirin inhibits irrerversibly.
Explain the proximity and orientation (catalysis by approximation) mechanism in enzyme catalysis.
When molecules are already in contact, as when they are in close proximity, the reaction doesn't require a collision of 2 molecules.
Explain the desolvation mechanism in enzyme catalysis.
The active site in enzyme catalysis moves the reactants away from H2O, effectively desolvating them.
What is the purpose of the desolvation mechanism?
By desolvating the reactants, you decrease the dielectric constant and increase the electrostatic interactions.
What does this do to the relationship between H2O and the reactants?
Reactive groups of reactants are protected from HO2, so HO2 doesn't compete with reactants. (HO2 won't react to give unwanted byproducts, e.g., by hydrolysis of some reactive intermediate in the reaction that was supposed to transfer its reactive group to another substrate.)
Explain the tight transition state binding mechanism in enzyme catalysis.
Because its "distortion" (electrostatic or structural) is "paid for" by . Free energy of transition state (peak of free energy barrier on reaction diagram) is lowered tighter bindthan of substrate.
Explain the induced fit mechanism in enzyme catalysis.
It deals with conformational change resulting from substrate binding.
Explain the general acid-base catalysis mechanism in enzyme catalysis.
A group that donates a proton (acts as a general acid) in catalysis has to then accept a proton (act as a general base) later in catalytic mechanism for in original form catalyst to be regenerated.
Explain the covalent catalysis mechanism of enzyme catalysis.
Rate acceleration by transient formation of a COVALENT catalyst-substrate bond is more reactive in next step in reaction, so covalent intermediate that step has lower activation energy than it would have for a non-covalent catalytic mechanism -- enzyme alters pathway to get to product.
Explain the metal ion catalysis mechanism of enzyme catalysis.
Metal ions can either be tightly bound or loosely bound. Functions of metal ions in catalysis include:
Why are peptide bonds kinetically stable in the absence of a catalyst, given that equilibrium lies far in
the direction of hydrolysis in 55.5 M H2O? (i.e., why is any specific reaction a slow reaction?)
Reason uncatalyzed reaction is so slow: partial double bond character of peptide bond makes carbonyl carbon much less reactive than carbonyl carbons in carboxylate esters.
What is the "job" of the catalyst (the protease), i.e., what group needs to be made more susceptible to nucleophilic attack?
Catalytic task of proteases is to make that normally unreactive carbonyl group more susceptible to nucleophilic attack by H2O.
Identify the nucleophile that attacks the carbonyl carbon in acylation; identify the nucleophile that
attacks the carbonyl carbon in deacylation.
The enzyme provides a potent nucleophile, a specific Ser OH group.

2nd substrate, H2O, is the nucleophile of deacylation
What is an acyl group?
An acyl group is R-C=O with another bond coming off the C
Describe the acyl-enzyme intermediate, including identifying the type of
bond attaching the acyl group to the enzyme (Is it an amide linkage? anhydride? ester? etc.) and how that acyl group relates to the structure of the original substrate.
It is a carboxylate ester. (ester bonded to the Ser residue on enzyme
Identify the leaving group coming from each of the tetrahedral intermediates as the intermediate breaks down.
The first tetrahedral intermedia'es leaving group is the O-C-C.
The second tetrahedral intermediate's leaving group is the carbonyl, becoming an oxyanion
State what is being acylated and deacylated in the chymotrypsin reaction
Explain the role of the "oxyanion hole" in the mechanism.
an area in the active site of serine proteases that binds the transition state particularly tightly
Describe which type(s) of general catalytic mechanisms are used by chymotrypsin, and how.
Compare (very briefly, just the “bottom line”) the overall 3-dimensional structures of chymotrypsin,
trypsin, and elastase, and compare the substrate binding specificities of those 3 enzymes, explaining
the relationship of the “specificity site/pocket” structure to the differences in substrate specificity.
How do 3 other classes of proteases (besides the serine proteases) generate nucleophiles potent enough
to attack a peptide carbonyl group?
Cys proteases: nucleophile is a His-activated Cys thiol (general base catalysis by His)
Asp proteases: nucleophile is HOH itself assisted by 2 Asp residues, general base catalysis by 1 Asp carboxyl group and orientation / polarization of substrate carbonyl by 2nd Asp residue
Metalloproteases: nucleophile is HOH assisted by binding to a metal (e.g. Zn2+) and by general base catalysis by some enzyme base group, e.g. Glu-COO-.
To which protease class does HIV protease belong?
It is a homodimer, where 2 identical subunits contribute an Asp to the active site
Describe the quaternary structure and
symmetry of the HIV protease and where in the quaternary structure the active site residues are
There are two flaps which close down after the substrate binds (induced fit). There are 2 catalytic Asp residues in the middle where the binding pocket resides. There is symmetry across the y-axis.
Describe the general features of the 3-dimensional structures of myoglobin and hemoglobin and the
similarities and differences between the 2 structures.
allosteric control
interaction with regulatory proteins
multiple forms of enzymes (isozymes)
reversible covalent modification
proteolytic activation
Explain the meaning of the “first committed step” in a metabolic pathway, and of the “rate-limiting step” in a metabolic pathway (which frequently turn out to be the same step), and explain the significance of those terms in regulation of “flow” of molecules through that pathway.
The most efficient step for the regulation of rate that should also be the step in pathway, controlling "flow" of matter to end product whose concentration you want to regulate regulation of the rate slowest
Define multimeric protein
An aggregate of more than one polypeptide chain
Define fractional saturation
It is the occupied sites / total sites for an enzyme
Define feedback inhibition
As endproduct of a metabolic pathway increases in concentration, cell needs to reduce rate of synthesis of that product
Briefly explain the allosteric regulation of ATCase, including its quaternary structure, its role in
metabolism, and how its activity is regulated by allosteric inhibition and activation. Include the
physiological rationale for the inhibition and activation.
Briefly explain the difference between the concerted model and the sequential model for an allosteric
concerted models shows all subunits being in the same conformation. In the sequential model, hybrid protein moleccules exist with some subunits in R states and some in T states. "hybrid" R/T molecules are possible in the sequential model (some subunits in R state, some in T state in same enzyme molecule).
Describe in general terms how cells carry out reversible covalent modification of enzymes, and by what means the modification would be removed.
Modification of catalytic or other properties of proteins by covalent attachment of a modifying group,
Name (“generic” names) the types of enzymes that catalyze phosphorylation.
Name the types of enzyme that catalyze dephosphorylation (a specific subclass of hydrolases) of proteins.
Dephosphorylation of enzymes is catalyzed by a specific (asubclassof ).proteinphosphatase hydrolases
What types of amino acid functional groups are generally the targets of phosphorylation?
The target is a protein, specifically a particular OH group on the protein. It can be with Serine/Threonine or Tyrosine.
Explain whether the dephosphorylation reaction is actually the chemical reverse of the phosphorylation reaction.
It is not the reverse of protein kinase-catalyzed phosphorylation.
Explain the regulation of protein kinase A (PKA) activity by cAMP, including quaternary structural changes in PKA triggered by cAMP binding.
PKA inactive form (without cAMP bound): inactive 2 catalytic subunits + 2 regulatory subunits.
What is a pseudosubstrate?
It has a slightly different AA sequence, specifically having an Ala in there instead of Ser or Thr. This prevents phosphorylation but allows binding of the PKA catalytic subunit, essentially blocking legitimate substrates from binding.
What does reciprocal regulation mean?
When multiple enzymes take part in opposing pathways, they are regulated against one another. Both enzymes end up being phosphorylated when the cell wants to get glucose out of storage. The ultimate result of this is that they aren’t both working in the cell at the same time.
What are the 2 enzymes involved in glycogen whos e activities are reciprocally regulated by phosphorylation/dephosphorylation?
The two enzymes involved are glycogen phosphorylase, and glycose synthase.
Briefly discuss the structure of calmodulin ( ± Ca2+), including the structure of the “EF hand” motif, and the ligand-binding properties of Ca2+-calmodulin, as an example of a regulatory protein.
The EF hand motif describes a helix-loop-helix unit common in the Ca2+ binding motif. The E helix is index finger, The F helix is the thumb, the Ca2+ is right above the curled middle finger.
Describe the general mechanism by which zymogens are activated to become active enzymes.
What occurs in chymotrypsinogen is there is a cleavage between Lys15 and Ile16. This generates a new alpha-amino group on Ile16. The new amino terminus of the new product chain turns inward and makes a new salt link that stabilizes the active conformation of chymotrypsin.
Discuss the protective mechanism that keeps prematurely activated pancreatic digestive enzymes inside the acinar cells from autodigesting the pancreas, and describe/name an example.
Small, very specific, very tight-binding inhibitor proteins inside cell inhibit any protease molecule that's accidentally activated inside the cell. An example would be pancreatic trypsin inhibitor, PTI.
Explain the term SERPIN and give examples of serpins that inhibit a) elastase, and b) thrombin.
Antithrombin III inhibits thrombin. Alpha-1 Antiproteinase keeps elastase inhibited.
How can a deficiency in alpha-antitrypsin contribute to emphyzema?
Elastase hydrolizes elastic fibers in alveolar walls of lungs causing emphysema. An inhibitor prevents this
Explain how a cascade of catalysts (e.g., in blood clotting) results in amplification of a signal.
Suppose 1 signalling molecule triggers activation of one molecule of Enzyme 1.
Describe the covalent modification of some of the clotting factors that requires the participation of vitamin K.
Vitamin K is required for activity of glutamate carboxylase.
What is the structure of the modified amino acid R group, and what is the 3-letter abbreviation of the modified residue?
The structure of the modified amino acid is called gamma-carboxyglutamate. The 3-letter abbreviation is GLA.
What ion is bound by the modified amino acid residues?
Briefly discuss how clots are confined to the area of injury, including the role of heparin and serpins such as antithrombin III.
Clots are confined to the area of injury through: dilution of blood flow, removal by the liver, protease degradation, and binding to specific inhibitor proteins.
One small protein inhibitor in plasma is antithrombin III. It binds thrombin and elastase. By itself, it binds the clotting factors very weakly.
Heparin is a negatively charged polysaccharide secreted by many cells. It binds to antithrombin III, producing a conformational change that increases the binding affinity of antithrombin for the serine proteases it inhibits. In the absence of heparin, antithrombin III binds very slowly to the clotting factors.
Describe how fibrinolysis (clot breakdown) is achieved.
It is achieved by proteolysis by plasmin, another serine protease that hydrolyzes peptide bonds in the fibrin clots.
What is TPA?
TPA is tissue-type plasminogen activator. It also has a fibrin-binding domain that targets it to fibrin clots, where it encounters plasminogen.