What is abzymes?

An abzyme (from antibody and enzyme), also called catmab (from catalytic monoclonal antibody), and
most often called catalytic antibody, is a monoclonal antibody with catalytic activity. Abzymes are usually
raised in lab animals immunized against synthetic haptens, but some natural abzymes can be found in
normal humans (anti-vasoactive intestinal peptide autoantibodies) and in patients with autoimmune
diseases such as systemic lupus erythematosus, where they can bind to and hydrolyze DNA. To date
abzymes display only weak, modest catalytic activity and have not proved to be of any practical use. [1] They
are, however, subjects of considerable academic interest. Studying them has yielded important insights
into reaction mechanisms, enzyme structure and function, catalysis, and the immune system itself.

1. The hydrolysis of pyrophosphate to orthophosphate is important in driving forward biosynthetic reactions

such as the synthesis of DNA. This hydrolytic reaction is catalyzed in E. coli by a pyrophosphatase that
has a mass of 120kdal and consists of six identical subunits. Purified enzyme has a V max of 2800 units
per milligram of enzyme. For this enzyme, a unit activity is defined as the amount of enzyme that
hydrolyzes 10μmol of pyrophosphate in 15 minutes at 37oC under standard assay conditions.
a. How many moles of substrate are hydrolyzed per second per milligram of enzyme when the substrate
concentration is much greater than Km.
b. How many moles of active site are there in 1mg of enzyme? Assume that each subunit has one active
site.
c. What is the turnover number of the enzyme?
2. Penicillin is hydrolyzed and thereby rendered inactive by penicillinase, an enzyme present in some
resistant bacteria. The mass of this enzyme in Staphylococcus aureus is 29.6kdal. The amount of
penicillin hydrolyzed in 1 minute in a 10-ml solution containing 10-9 g of purified penicillinase was
measured as a function of the concentration of penicillin. Assume that the concentration of penicillin
does not change appreciably during the assay.
[Penicillin] Amount hydrolyzed (moles)
0.1 x 10-5M 0.11 x 10-9M
0.3 x 10-5M 0.25 x 10-9M
0.5 x 10 M
-5 0.34 x 10-9M
1.0 x 10-5M 0.45 x 10-9M
3.0 x 10-5M 0.58 x 10-9M
5.0 x 10-5M 0.61 x 10-9M
a. Make a 1/V versus 1/[S] plot of these data. Does penicillinase appear to obey Michaelis-Menten kinetics?
If so, what is the value of Km?
b. What is the value of Vmax?
c. What is the turnover number of penicillinase under these experimental conditions? Assume one active
site per enzyme molecule.
3. The kinetics of an enzyme are measured as a function of substrate concentration in the presence and
absence of 2x10-3M inhibitor (I).

Velocity (μmol/min)
[S]
No inhibitor Inhibitor
0.3 x
10-5M 10.4 4.1
0.5 x 10 M
-5 14.5 6.4
1.0 x 10 M
-5 22.5 11.3
3.0 x 10-5M 33.8 22.6
9.0 x 10 M
-5 40.5 33.8
a. What are the values of the Vmax and KM in the absence of inhibitor? in its presence?
b. What type of inhibition is this?
c. What is the binding constant of this inhibitor?
d. If [S] = 1 x 10-5 M and [I] =2 x 10-3 M, what fraction of the enzyme molecules have a bound substrate? a
bound inhibitor?
e. If [S] = 3 x 10-5 M, what fraction of the enzyme molecules have a bound substrate in the presence of 2
x 10-3 M inhibitor? Compare this ratio with the ratio of the reaction velocities under the same conditions.
4. The kinetics of the enzyme considered in problem 3 are measured in the presence of a different inhibitor.
The concentration of this inhibitor is 100 μM.
[S] Velocity (μmol/minute)
(μM) No Inhibitor Inhibitor
3 10.4 2.1
5 14.5 2.9
10 22.5 4.5
30 33.8 6.8
90 40.5 8.1
(a) What are the values of Vmax and KM in the presence of this inhibitor? Compare them with those obtained
in problem 3.
(b) What type of inhibition is it?
(c) What is the dissociation constant of this inhibitor?
(d) If [S] = 30 μM, what fraction of the enzyme molecules have a bound substrate in the presence and in
the absence of 100 μM inhibitor?
5. The plot of 1/V versus 1/[S] is sometimes called a Line-Weaver Burk plot. Another way of expressing
the kinetic data is to plot V versus V/[S], which is known as an Eadie-Hofstee plot.
a. Rearrange the Michaelis-Menten equation to give V as a function of V/[S]?
b. What is the significance of the slope, the y-intercept, and the x-intercept in a plot of V versus V/[S]?
c. Make a sketch of a plot V versus V/[S] in the absence of an inhibitor, in the presence of a competitive
inhibitor, and in the presence of a non-competitive inhibitor.
6. For allosteric enzymes, low concentrations of competitive inhibitors frequently act as activators? (Hint:
Consider the analogy of CO-hemoglabin.)

7. The hormone progesterone contains two ketone groups. Little is known about the properties of the
receptor protein that recognizes progesterone. At pH 7, which amino acid side chains might form
hydrogen bonds with progesterone? (Assume that the side chains in the receptor proteins have the
same pKs as in the amino acids in the aqueous solution).
8. Suppose that two substrates A and B compete for an enzyme. Derive an expression relating the ratio of
the rates of utilization of A and B, VA and VB, to the concentrations of these substrates and their values
of k3 and KM. (Hint: express VA as a function of k3/ KM for substrate A and do the same for VB.) Is
specificity determined by KM alone?
9. A tenacious mutant. Suppose that a mutant enzyme binds a substrate 100-fold as tightly as does the
native enzyme. What is the effect of this mutation on catalytic rate if the binding of the transition state
is unaffected?

10. Uncompetitive inhibition. The following reaction represents the mechanism of action of an
uncompetitive inhibitor.

(a) Draw a standard Michaelis-Menten curve in the absence and in the presence of increasing amounts of
inhibitor. Repeat for a double-reciprocal plot.
(b) Explain the results obtained in part a.
11. For an enzyme that follows simple Michaelis-Menten kinetics, what is the value of Vmax if V0 is equal to
1 μmol/minute at 1/10 KM?

12. For a one-substrate, enzyme-catalyzed reaction, double-reciprocal plots were determined for three
different enzyme concentrations. Which of the following three families of curve would you expect to be
obtained? Explain.
13. A simple Michaelis-Menten enzyme, in the absence of any inhibitor, displayed the following kinetic
behavior. The expected value of Vmax is shown on the y-axis.

(a) Draw a double-reciprocal plot that corresponds to the velocity-versus-substrate curve.

(b) Provide an explanation for the kinetic results.
14. In the conversion of A into D in the following biochemical pathway, enzymes E A, EB, and EC have
the KM values indicated under each enzyme. If all of the substrates and products are present at a
concentration of 10-4 M, which step will be rate limiting and why?
15. The effect of pH on the activity of an enzyme was examined. At its active site, the enzyme has an
ionizable group that must be negatively charged for substrate binding and catalysis to take place. The
ionizable group has a pKa of 6.0. The substrate is positively charged throughout the pH range of the

experiment.
(a) Draw the V0-versus-pH curve when the substrate concentration is much greater than the enzyme KM.
(b) Draw the V0-versus-pH curve when the substrate concentration is much less than the enzyme KM.
(c) At which pH will the velocity equal one-half of the maximal velocity attainable under these conditions?
16. Pyridoxal phosphate (PLP) is a coenzyme for the enzyme ornithine aminotransferase. The enzyme
was purified from cells grown in PLP-deficient media as well as from cells grown in media that contained
pyridoxal phosphate. The stability of the enzyme was then measured by incubating the enzyme at
37°C and assaying for the amount of enzyme activity remaining. The following results were obtained.

(a) Why does the amount of active enzyme decrease with the time of incubation?
(b) Why does the amount of enzyme from the PLP-deficient cells decline more rapidly?
 Technological Institute of the Philippines-Manila
363 P. Casal St. Quiapo, Manila

HW in CHE 503-Biochemical Engineering

Submitted by: Yabut, Raymond Joseph B.

Submitted to: Engr. Michael Francis Sy

Date Submitted: August 31, 2018