Exam 2 - questions Flashcards
- Describe how you would determine the Ka (association constant) for a ligand and a protein.
An experiment would be carried out in which a fixed amount of the protein is incubated with
varying amounts of ligand (long enough to reach equilibrium). The fraction of protein molecules that
have a molecule of ligand bound is then determined. A plot of this fraction () vs. ligand concentration
[L] should yield a hyperbola. The value of [L] when = 0.5 is equal to 1/Ka
For the binding of a ligand to a protein, what is the relationship between the Ka (association
constant), the Kd (dissociation constant), and the affinity of the protein for the ligand?
Ka = 1/Kd. The larger the Ka (and hence the smaller the Kd), the higher the affinity of the protein
for the ligand.
What fraction of ligand binding sites are occupied () when [ligand] = Kd? Show your work.
From equation (5 - 8) on page 156 : = [Ligand]/ [Ligand] + Kd thus, when [Ligand] = Kd , the equation becomes : = Kd 1 Kd + Kd 2
Describe briefly the two principal models for the cooperative binding of ligands to proteins with
multiple binding sites
In the concerted model, binding of a ligand to one site on one subunit results in an allosteric
effect that converts all of the remaining subunits to the high-affinity conformation. As a result, all of
the subunits are either in the low- or high-affinity conformation. In the sequential model, each
subunit is changed individually to the high affinity conformation. As a result, there are many possible
combinations of low- and high-affinity subunits.
Describe briefly the basic structure of an IgG protein molecule.
An IgG protein contains two copies of a large polypeptide (heavy chain) and two copies of a small polypeptide (light chain). structure contributes significantly to the tertiary structure of domains of both chains. Disulfide bonds link the heavy chains to one another and to the light chains. The chains are arranged in a Y-shaped structure where the two arms are linked to the base by a protease sensitive (“hinge”) region
What is the concept of “induced fit” as it applies to antigen-antibody binding?
The conformations of the antigen and antigen-binding site of the antibody are influenced by
each other and change as binding occurs. These conformational changes increase the chemical
complementarity of the sites and result in tighter binding.
Describe how immunoaffinity chromatography is performed.
The specific antibody is covalently attached to an inert supporting material, which is then
packed into a chromatography column. The protein solution is passed through the column slowly;
most proteins pass directly through, but those for which the antibody has specific affinity are
adsorbed. They can subsequently be eluted by a buffer of low pH, a salt solution, or some other agent
that breaks the antibody-antigen association.
What properties of antibodies make them useful biochemical reagents? Describe one biochemical
application of antibodies (with more than just the name of the technique)
The important properties are the high specificity of protein recognition, and the high affinity of
the antibody-antigen association. These make possible immunoaffinity chromatography,
immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), and immunoblotting, all of
which are described on pp. 173-175.
Draw and label a reaction coordinate diagram for an uncatalyzed reaction, S P, and the same
reaction catalyzed by an enzyme, E
See Fig. 6-3, p. 187.
Write out the equation that describes the mechanism for enzyme action used as a model by
Michaelis and Menten. List the important assumptions used by Michaelis and Menten to derive a
rate equation for this reaction.
The two equations are
k1 k2
E + ES E + P
k-1 k-2
One assumption is that [P] = 0, so that the rate of the reaction depends exclusively on the breakdown
of ES and is not influenced by the reverse reaction; that is, k-2 can be ignored and V0 = k2 [ES]. This
condition is possible only if early reaction times are measured; the velocity, therefore, is an initial
velocity. A second assumption is that the rate of ES formation equals the rate of ES breakdown; in
other words, the reaction is at a steady state. A third assumption is [S]»_space; [Et], so that total [S],
which equals free substrate and enzyme-bound substrate, is essentially equal to [S].
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?
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.
Give the Michaelis-Menten equation and define each term in it. Does this equation apply to all
enzymes? If not, to which kind of enzymes it doesn’t apply?
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.
Methanol (wood alcohol) is highly toxic because it is converted to formaldehyde in a reaction
catalyzed by the enzyme alcohol dehydrogenase:
NAD+ + methanol NADH + H+ + formaldehyde
Part of the medical treatment for methanol poisoning is to administer ethanol (ethyl alcohol) in
amounts large enough to cause intoxication under normal circumstances. Explain this in terms of
what you know about examples of enzymatic reactions.
Ethanol is a structural analog of methanol, and competes with methanol for the binding site of
alcohol dehydrogenase, slowing the conversion of methanol to formaldehyde, and allowing its
clearance by the kidneys. The effect of ethanol is that of a competitive inhibitor.
What is a zymogen (proenzyme)? Explain briefly with an example
A zymogen is an inactive form of an enzyme that is activated by one or more proteolytic cleavages
in its sequence. Chymotrypsinogen, trypsinogen, and proelastase are all zymogens, becoming
chymotrypsin, trypsin, and elastase, respectively, after proper cleavage.
This compound is L-glyceraldehyde. Draw a stereochemically correct representation of C-1 and C2 of D-glucose. CHO | HO—C—H | CH2OH
In D-glucose, the positions of the —H and —OH on C-2 are the reverse of those for C-2 of Lglyceraldehyde. (Compare Fig. 7-1, p. 236, with Fig. 7-2, p. 236.)
