THE INTERNATIONAL UNIVERSITY (IU) – VIETNAM NATIONAL UNIVERSITY – HCMC

FINAL EXAMINATION
Date: ......................................
Duration: 90 minutes
Student ID: .................................. Name:................................................

SUBJECT: Enzymology
Dean of School of Biotechnology Lecturer
Signature: Signature:

Full name: Full name: Dr. Nguyễn Tấn Khôi

GENERAL INSTRUCTION(S)

1 This is an open note examination; NO TEXTBOOK, NO INTERNET! Laptops are

allowed for computational purposes.
2. ABSOLUTELY NO form of communication is allowed during the exam.
3. No dictionary is allowed.

GOOD LUCK!

QUESTION

1. State the rapid equilibrium and steady state hypotheses. Compare these two as
well as list out the pros and cons of each. (20 pts)
2. Now you study enzyme inhibition by measuring enzyme kinetics in the presence
of 10 mM of inhibitor A or inhibitor B (separately). The Lineweaver-Burk plots in
the presence of these inhibitors are indicated by “+A” or “+B” in the Figure
below.
 a) (15 pts) From these data determine the type of inhibition for: A and B

b) (15 pts) Which of the two inhibitors is more efficient at high substrate
concentrations? At low substrate concentrations? Show you reasoning.

3. (50 pts) Studies of binding at equilibrium were then performed with stable
substrate and products, namely NADPH, NADP+ and catechin. For this purpose,
we selected the chromatographic method of Hummel and Dreyer which had the
advantage over equilibrium dialysis to be applicable to non-radiolabeled but
chromophoric ligands, in the hypothesis of fast equilibria. We used 100 mM Tris
pH 7 plus 75 mM NaCl because of a marked tendency of the enzyme to undergo
aggregation within 10 min in other buffers such as HEPES, Bis–Tris/Tricine or
phosphate. Although it would have been preferable to work at pH 7.5, this
problem of aggregation could not be eliminated at this pH with the high enzyme
concentrations which were required for such experiments. The analysis of
NADPH binding to the free enzyme is summarized in Figure below. In these
experiments, the absorbance wavelength of the detector was fixed at 340 nm and
we used a molar extinction coefficient of 6220 M-1 cm-1 for NADPH. The eluent
contains a fixed concentration of NADPH, and the 340nm absorbance provided
by the detector is set to zero in the absence of injected sample. When a sample of
free enzyme which has been dissolved in the NADPH-free buffer is injected
without co-injection of NADPH, a negative peak or trough appears at the
retention time of NADPH. This only reflects the lack of NADPH in the sample.
When increasing concentrations of NADPH are co-injected with the enzyme, the
area of the negative peak linearly decreases and eventually becomes positive
above a threshold concentration of co-injected NADPH (fig A). This threshold
 concentration can be accurately estimated by means of linear regression (see
internal calibration of Fig. B), and it is equal to the sum of free and bound
NADPH at equilibrium.

Figure: Hummel and Dreyer analysis of NADPH binding to the free enzyme. (A) All
chromatograms were obtained upon injection of 100 µl of 32.8 µM ANR (1.14 gl -1) with
50 µM NADPH fixed in the eluent; chromatograms (a–e) were obtained upon co-
injection of increasing concentrations of NADPH (0, 25, 50, 100 and 200 µM); peaks 1
and 2 appear at the retention time of enzyme and free NADPH, respectively. (B)
Graphical estimation of the threshold concentration of co-injected NADPH that is
required for peak 2 inversion. (C) Scatchard plot; f, free; b, bound.

a. Perform the linear regression to determine the estimate of this threshold at its 95%
confidence interval.
b. At this threshold concentration of the substrate, determine the binding constant at
equilibrium.
c. Recall the Scatchard plot you learnt in Biophysical Chemistry, does figure C
imply that NADPH acts as an inhibitor?

Good luck!