BCH 4053 PRE-TEST 2 GROUP NAME _____________________
June 19, 1996
This test is take-home and open book, and it is
intended that all members of the group contribute to
completing it. Only one copy is to be submitted by
the group, and all members who participated should
sign their names below. Test is due by 1:30 pm on
Monday, June 24.
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Points
(23) 1. A biochemist studying the properties of a newly isolated metabolic
enzyme obtains the following rate data during kinetic experiments in
the absence and presence of two different inhibitors, A and B, one a
substrate analogue.
[S] v v v
(M x 104) æmol/min æmol/min æmol/min
5.0 1.25 0.74 0.48
2.5 0.87 0.45 0.33
1.7 0.67 0.32 0.25
1.2 0.54 0.25 0.20
1.0 0.45 0.21 0.17
Inhibitor A concentration is 5 x 10-4 M; that of B is 3.2 x 10-6 M.
a. Determine Vmax and KM for this enzyme using the Lineweaver-Burk
reciprocal plot. Plot the inhibitor data on the same graph.
(Note: Pick your axes and scales carefully so that the lines
may be extrapolated to the negative x intercept. It would be a
good idea to draw the graph on scratch graph paper first, then
do a clean finished copy.)
b. Classify the inhibitors as competitive, not-competitive, non-
competitive, or uncompetitive. Which one is likely to be the
substrate analogue?
c. Plot the data for the uninhibited reaction on a separate graph
using the Eadie-Hofstee plot (v plotted versus v/S). Determine
Vmax and Km for the uninhibited reaction from this graph, and
compare with part a.
(Show your calculations on a blank sheet of paper, and plot your
graphs on the graph paper, and attach them to the exam. Put
your group name on the sheet and the graph paper.)
(12) 2. Following are three models for reversible inhibition of a simple
one-substrate enzyme reaction, three rate laws which are derived from
the models, expressed in reciprocal form, and four terms describing
types of reversible inhibition. For the graphs which follow,
indicate in the blank below the graph the model (a, b, or c), the
rate law (d, e, or f), and the term(s) (g, h, i, or j) which apply to
that graph.
(a) E + S þ ES E + P (b) E + S þ ES E + P
E + I þ EI ES + I þ ESI
(c) E + S þ ES E + P where (for a, b, and c):
E + I þ EI KI = [E][I]/[EI]
ES + I þ ESI K'I = [ES][I]/[ESI]
(d) 1/v = (1/Vm)(1 + I/K'I) + (Km/Vm)(1/S)
(e) 1/v = (1/Vm)(1 + I/K'I) + (Km/Vm)(1/S)(1 + I/KI)
(f) 1/v = (1/Vm) + (Km/Vm)(1/S)(1 + I/KI)
(g) competitive
(h) not-competitive
(i) non-competitive
(j) un-competitive
³ ³
³ lines ³
1/v ³ intersect on 1/v ³ parallel
³ x axis ³ lines
³ ³
³ ³
ÄÄÄÄÄÁÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ÄÄÄÄÄÁÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
1/S 1/S
_________________ ________________
³ ³
³ lines ³ lines
1/v ³ intersect on 1/v ³ intersect off
³ y axis ³ axes
³ ³
³ ³
ÄÄÄÄÄÄÁÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ÄÄÄÄÄÁÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
1/S 1/S
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(12) 3. Subtilisin (MW = 27,600) is a bacterial protease that can catalyze
hydrolysis of certain amino acid esters and amides. For the
synthetic substrate N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt),
subtilisin exhibits Km and kcat (or k2 as used in lecture) values of
0.15 M and 550 s-1, respectively.
(a) What is the Vmax for the hydrolysis of Ac-Tyr-OEt when the
subtilisin concentration is 0.4 mg ml-1?
(b) Indole is a competitive inhibitor of subtilisin with a Ki of
0.05 M. What is the Vmax for Ac-Tyr-OEt hydrolysis by
0.40 mg ml-1 subtilisin in the presence of 6.25 mM indole?
(c) What is the velocity (v) when 0.40 mg ml-1 subtilisin is
incubated with 0.25 M Ac-Tyr-OEt?
(9) 4. Classify the following fatty acids as þ-9, þ-7, þ-6, or þ-3.
9-C18:1 ________ 5,8,11,14-C20:4 _________ 9-C16:1 _______
9,12-C18:2 _______ 9,12,15-C18:3 _________ 8,11,14-C20:3 _______
(8) 5. Circle the following lipids which are negatively charged at pH 6.
phosphatidyl choline phosphatidyl serine
phosphatidyl glycerol cholesterol
sphingomyelin palmitic acid
phosphatidic acid phosphatidyl inositol
(12) 6. The mechanism of chymotrypsin illustrates several of the factors that
are believed to contribute to the rate acceleration obtained by
enzymes. Describe each of the following aspects of the chymotrypsin
mechanism.
(a) A reaction model that shows ping-pong kinetics. (i.e., identify
the components of the ping-pong mechanism, figure 8-13b, page
219).
(b) Transition state stabilization by bonds formed between the
enzyme and the transition state that are not found in the
binding of substrate or product.
(c) Acid-base catalysis mediated through a "catalytic triad".
Describe how the triad assists in the formation of the
covalently bound intermediate.
(d) Substrate specificity provided by the nature of the substrate
binding site. (Explain how chymotrypsin differs from trypsin in
the binding site.)
(9) 7. Draw the structures of the products from the following reactions:
(a) mild acid hydrolysis of choline plasmalogen.
(b) mild base hydrolysis of phosphatidyl serine
(c) phospholipase A hydrolysis of phosphatidyl glycerol
(6) 8. Describe three types of lipid anchors that attach some proteins to
membranes.
(9) 9. Distinguish between integral and peripheral membrane proteins in
terms of
(a) types of solutions used to extract them from membranes.
(b) forces by which they are attached to membranes.
(c) membrane location in the fluid mosaic model.