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2 Apr 2019

A 5 00Î¼l reaction co ntaining 0.5 pmol of an enzyme following Michaelis â Menten kinetics with saturating substrate concentration is studied . For this enzyme k1 = 1 x 10 9 M - 1 s - 1 , k - 1 = 2 x 10 3 s - 1 , and k2 = 1 x 10 5 s - 1 .

a . What is the Vmax for the enzyme considering that it has only one substrate binding site ? b. What are the values of Km and Ks? Is this a rapid equilibrium enzyme or does it follow steady state kinetics? Explain why .

c . What is the substrate concentration needed to achieve half maximal velocity? Explain the basis for your answer.

d . If the substrate concentration in part c is doubled , how many fold will the initial velocity increase?

e . What is the Km i f the concentration of the enzyme is doubled? f . What is the efficiency of this enzyme?

g. Would this enzyme be considered a perfect enzyme? Explain.

h . If 0.25 pmol of an irreversible or classic non - competitive inhibitor is added before starting the reaction. What is the resulting Km for the enzyme?

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2 Apr 2019

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Related questions

In a 50Âµl reaction containing 0.25 pmol of a Michaelis enzyme with Km = 4.0 x 10-7 M and substrate concentration of 2 x 10-7 M, product was formed at an initial velocity of 5.0 x 10-5 Mmin-1 .

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describe the definitions of substrate, enzyme active site and its general characteristics, and apoand

holo-enzymes.

Â· describe the fact that enzyme catalysis is specific in terms of the type of reaction and the exact

substrate structure.

Â· Explain the difference in the âlock-and-keyâ model and the induced fit model of enzyme

substrate interaction.

Â· describe that reaction rate constant thus reaction rate is dependent on the activation energy.

Â· describe the relationship between the energy of the transition state and the activation energy.

Â· describe the relative binding affinity of a substrate and its transition state to the enzyme.

Â· Explain how enzymes speed up reaction rate, i.e., how do enzymes lower the activation

energy. Note: Factors other than preferential stabilization of transition state also contribute to increased

reaction rate. They include 1) enzymes placing one substrate (reactant) next to the other substrate (reactant)

so that the reaction is no longer dependent on the collision rate of the two substrates; 2) enzymes sequester the

substrate(s), minimizing solvent competition thus speeding up the reaction; 3) in enzyme complexes with each

enzyme catalyzing one step in a multi-step reaction sequence, the product of one reaction is fed to the active site

of another enzyme for the next step without the product diffusing away from the enzyme complex thereby

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Â· describe the Michaelis-Menten equation relating the initial velocity to total substrate

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Â· describe the assumptions that went into the derivation of the Michaelis-Menten equation. ([E]

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Â· describe the relationship between Vmax and kcat and the fact that kcat is a constant for a given

enzyme and substrate at a given temperature and solution condition; whereas Vmax can differ

for a given enzyme and substrate at a given temperature and solution condition, depending

on the enzyme concentration.

Â· describe the definition of Kd (equilibrium dissociation constant for ES into E and S).

Â· describe the relationship between Kd and the affinity of substrate for the enzyme, i.e., the

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Â· describe the relationship between Kd and Km, i.e., in general, the higher the Kd, the higher theKm.

Â· Use the Michaelis-Menten equation and the relationship between Vmax and kcat to do

calculations.

Â· describe the other names for the catalytic rate constant, kcat (i.e., the turn over number; also

called krelease) and why it is so called (kcat reports on per unit time per enzyme molecule the

number of product molecules formed/released, thus the name turnover number or krelease).

Â· Determine Vmax and Km using both the Michaelis-Menten plot (initial velocity versus [S]) and

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[S].

Â· Draw the Michaelis-Menten plot AND the double reciprocal plot (Lineweaver Burk), given

the Vmax and Km values.

Â· describe the fact that the term kcat/Km is the best parameter for measuring the catalytic

capability (efficiency) of an enzyme.

Â· describe the existence of irreversible enzyme inhibition and the general mechanisms of

irreversible inhibition.

Â· describe the different types of reversible inhibitions, namely the competitive, uncompetitive,

and mixed inhibitions (noncompetitive inhibition is a special case in which the inhibitor

binds E and ES with equal affinity therefore Î± is equal to Î±â â Km is not affected).

Â· Describe each type of inhibition in terms of inhibitor binding site (active site or other than

the active site), how the inhibitor affects vmax and Km and the quantitative relationship of

each parameter with and without the inhibitor present and their relationship with [I] and KI

or KI

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velocity versus total substrate concentration), i.e. draw the curves with and without the

competitive inhibitor.

Â· Show the characteristics of each type of inhibition on a double reciprocal plot (LineweaverBurk

Plot) by drawing the lines with and without the competitive inhibitor.

Â· Given the Michaelis-Menten or double reciprocal plot with and without the inhibitor,

determine the type of inhibitor.

Â· describe the fact that transition state analogs are potent inhibitors.

Â· Explain why transition state analogs are potent inhibitors.

Â· describe that not all enzymes obey Michaelis-Menten kinetics.

Â· describe situations when an enzyme will not obey Michaelis-Menten kinetics (when the enzyme

catalyzes reactions with multiple substrates or when the enzyme is allosteric, i.e. binding of

the substrate at one active site affects the other active site(s) for the same substrate).

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and difference.

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Â· State the catalytic role of each of the catalytic residues and how they accomplish that.

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Â· Draw the transition state structure for the formation of the acyl-enzyme intermediate and

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Â· describe the steps in the regeneration of chymotrypsin (deacylation), the substrates and

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stabilized by the enzyme.

Â· Explain what general acid and base catalysis refers to and what groups act as the general acid

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Â· Explain the basis for the substrate specificity of chymotrypsin (consider the composition of

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residues)

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nucleophile, which can attack the carbonyl carbon of the peptide bond).

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(specific methods of catalysis utilized by these enzymes; general description of overall

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Â· describe the definition of feedback inhibition (mechanism of cellular regulation that regards an

enzyme that catalyzes the production of a given substance that then inhibits the cell when

concentration of that substance has reached an appropriate level â this balances/regulates

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proteins.

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Ã Draw a curve for the initial reaction velocity versus substrate concentration.

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Ã Draw on the same graph above a v0 vs. [S] curve for the enzyme in the

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upon substrate binding. Comment on the shape of this curve.

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Ã When an activator is present for such an allosteric enzyme, the v0 versus.

Ã describe the relationship between feedback inhibition and allosteric regulation

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this covalent modification.

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An enzyme catalyzed reaction has a KM of 5 mM and a Vmax of 35 nM s-1. What is the reaction velocity when the substrate concentration is: (a) 0.25mM (b) 6.5mM (c) 15mM For the same enzyme the total enzyme concentration is [ET] = 15 pM (nM/1000). What is the kcat (turnover number) and kcat/KM (the apparent second order rate constant of the enzymatic reaction)? What is this a measure of?