Enzymes Problem SetEnzymes Problem SetKineticsInhibition

SubjectiveSubjective1) For the reaction E + S ® ES ® P the Michaelis-Menten constant, Km, is actually a summary of three terms. What are they? How is Km determined graphically?   2) Give the Michaelis-Menten equation and define each term in it. Does this equation apply to all enzymes? If not, to which kind does it not apply?  Ans:  3) Why does pH affect the activity of an enzyme?  Ans: The state of ionization of several amino acid side chains is affected by pH, and the activity of many enzymes requires that certain of the amino acid residue side chains be in a specific ionization state.

Subjective SolutionsSubjective SolutionsAns: Km = (k2 + k-1)/ k1, where k-1 and k1 are the rate constants for the breakdown and association, respectively, of the ES complex and k2 is the rate constant for the breakdown of ES to form E + P. Km can be determined graphically on a plot of V0 vs. [S] by finding the [S] at which V0 = 1/2 Vmax. More conveniently, on a double-reciprocal plot, the x-axis intercept = –1/ Km.   Ans: The Michaelis-Menten equation is: V0 = Vmax [S]/( Km + [S]), in which V0 is the initial velocity at any given concentration of S, Vmax is the velocity when all enzyme molecules are saturated with S, [S] is the concentration of S, and Km is a constant characteristic for the enzyme. This equation does not apply to enzymes that display sigmoidal V0 vs. [S] curves, but only to those giving hyperbolic kinetic plots.  Ans: The state of ionization of several amino acid side chains is affected by pH, and the activity of many enzymes requires that certain of the amino acid residue side chains be in a specific ionization state.

Hexokinase and Hexokinase and GlucokinaseGlucokinase Enzymes can use Km and Vmax to regulate

critical cellular processes, or even to affect tissue distribution of various metabolites.

An example of this can be seen in the two similar enzymes hexokinase and glucokinase, which both convert glucose to glucose-6-phosphate, an intermediate in the glucose oxidation pathway which cannot exit the cell.

Once glucose enters a cell and is converted to glucose-6-phosphate, it is "trapped" in that cell, and can thus only be used in the metabolism of that particular cell.

This is of particular importance in the brain, which cannot synthesize glucose, and thus has an absolute requirement for glucose from the blood.

Hexokinase is found in the brain and in skeletal muscle, and is a regulatory enzyme (i.e. it is inhibited by high concentrations of its product). It also has a higher affinity for glucose, with a Km value of 5 X 10(-5) M.

By contrast, glucokinase is found in the liver, and is absent in brain and muscle. It is non-regulatory, and has a lower affinity for glucose (Km = 2 X 10(-2) M). For comparison, note that the normal blood concentration of glucose falls in between these two values, as shown in the graph above.

At normal blood glucose, hexokinase is operating at near Vmax, ensuring that the brain gets an ample supply of glucose.

Glucokinase is operating far below its Vmax under these conditions.

If blood glucose rises significantly, hexokinase can speed up slightly, but glucokinase speeds up dramatically.

In this way, the excess blood glucose is taken up by the liver, and converted to glycogen and fat.

If blood glucose falls below normal, hexokinase is still operating near Vmax, while glucokinase is essentially inactive.

In this way, a constant supply of glucose is ensured for the brain at all times.

ProblemProblemThe Km for the reaction of chymotrypsin with N-acetylvaline ethyl ester is 8.8 x 10-2 M, and the Km for the reaction of chymtrypsin with N-acetyltyrosine ethyl ester is 6.6 x 10-4 M.

a. Which substrate has the higher apparent affinity for the enzyme?

b. Which substrate is likely to give a higher value for Vmax?

Classes of EnzymesClasses of Enzymes Oxidoreductases are enzymes which catalyze oxidation or

reduction reactions. An example is the reductase (dehydrogenase) alcohol dehydrogenase, a human enzyme which converts ethanol to acetaldehyde. A second enzyme known as aldehyde dehydrogenase then converts acetaldehyde to acetyl CoA. Oxidoreductases often require a cofactor, in this case NAD+, which accepts the hydrogens released during oxidation.

Oxidases are so named only if oxygen is used as an acceptor molecule. An example is glucose oxidase, which converts sugars such as glucose to the corresponding gluconic acid.

Transferases are enzymes which transfer a functional group from a donor molecule to an acceptor molecule. A common transferase is a methyltransferase, which transfers a methyl group from S-adenosylmethionine to some acceptor. The example shown below is catechol-O-methyltransferase, which is an enzyme involved in the catabolism of the neurotransmittwers epinephrine and norepinephrine

A transaminase takes one amino acid and one alpha keto acid, and converts it to a second amino acid and a corresponding alpha keto acid, thus apparently transferring an amino functional group from one molecule to another. This allows for the interconversion of certain amino acids, and also allows amino acids to enter into glucose metabolism

Lyases catalyze the cleavage of C-C, C-O and C-N bonds by a means other than hydrolysis. An example is the important enzyme DOPA decarboxylase, which is a critical step in the synthesis of the biogenic amines epinephrine and norepinephrine.

Isomerases are simply enzymes which catalyze intramolecular rearrangements. Epimerases and racemases are examples of this class.

Ligases catalyze the formation of C-O, C-S, C-N or C-C bonds coupled with the hydrolysis of the pyrophosphate bond of ATP. The phosphate may or may not be covalently bound to the product.

Salicylate (aspirin) inhibits the catalytic action of glutamate dehydrogenase. Plot the data two ways: 1)v vs. [S] and 2) 1/v vs 1/[S] on graph paper. Estimate the Vmax and Km in the presence and absence of this inhibitor. How well do the estimates agree from the two plots. From the data, can you determine the type of inhibition (competitive or non-competitive)?

An Inhibitor of glutamate dehydrogenase was tested. Do the same calculations on this inhibitor. From the data, can you determine the type of inhibition (competitive or non-competitive)?How well do they agree?