MIT Department of Biology
7.013: Introductory Biology - Spring 2005
Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. Claudette Gardel
S’05 7.013 Section Problem- Enzymes/Protein Structure
There is a class of related enzymes called serine proteases that all use the same mechanism to
cleave peptide bonds. Each member of this family cleaves protein substrates at a different
location – that is, each enzyme cleaves protein substrates after a different specific amino acid.
Formally, they catalyze the following reaction.
A-B-C-D-X-E-F-G-H + H2O --------------->A-B-C-D-X
protein substrate
protein fragment
+
E-F-G-H
protein fragment
Where A through H are any amino acids, and X is one of the specific amino acids uniquely
recognized by the enzyme. If this process repeats again and again, a large substrate protein will
be completely degraded into small peptide fragments.
Each member of the protease family cleaves after a specific amino acid on the substrate because
that particular amino acid fits in the binding pocket of the protease enzyme.
a) One of these protease enzymes, trypsin, cleaves after lys or arg. What would be the
product(s) if each of the following peptide molecules were treated with trypsin?
i) leu-thr-phe-ala-ser
ii) trp-tyr-lys-ala-phe
iii) lys-arg-lys-arg
The structures of three hypothetical proteases binding pockets are shown below (the actual
enzyme is much larger than the binding pocket). If the amino acid side chain on the substrate
binds well to the binding or “recognition pocket” the substrate will bind and subsequently cleaved.
GENERIC ENZYME:
ACTIVE SITE -
peptide bond cleavage
gly
gly
gly
RECOGNITION POCKET specific substrate regognition
protease A
gly
gly
asp
protease B
ile
val
phe
protease C
b) The specificity of each protease (the amino acids that it likes to cut after) are listed
below.
Match the enzyme with the specificity & explain.
i) lysine, arginine
ii) phenylalanine, tryptophan, tyrosine
iii) glycine, alanine
AA sidechains, 7.013 CG 2004, 1
c) How might you design a similar enzyme to cleave after aspartic acid?
d) Speculate on the effect of changing the aspartic acid in protease B to a glutamic acid.
e) There are three amino acids required for the active site to function and three amino
acids involved in substrate recognition – why then do these enzymes typically contain
more than 200 amino acids?
f) Suppose you make a solution of protease A. A small sample taken when the solution was
made (time = 0) is capable of cleaving 100 mmol of protein substrate per minute. This
rate of substrate cleavage is called the “activity” of the enzyme.
At regular intervals over the next few hours as the solution of protease A stands at room
temperature, you remove small samples and measure the activity (the rate at which a
substrate protein is cleaved.).
You find that the activity of the enzyme drops rapidly as time passes.
However, interestingly, if when you made your solution of protease A, you added a large
amount of casein (a protein found in milk that has no enzymatic activity), the protease
loses its activity much more slowly. These data are sketched below:
activity
100
protease A + casein
protease A alone
0
0
time
Explain these observations.
AA sidechains, 7.013 CG 2004, 2
g) If you monitor the reaction of any of these proteases as they degrade a protein, you
observe that the protease does not cut all the recognition sites in the substrate at oncethe recognition sites on the surface of the substrate protein are the first to be cut.
Explain.
h) How might your answer to parts (f) and (g) be combined to design a long lasting
protease A molecule?
i) Normally, protease A is synthesized as an inactive precursor, “zymogen A”. Zymogen A
can be cleaved by protease A to form active protease A.
catalyzed by protease A
zymogen A + H2O -----------------------------> protease A + small
peptide
(inactive)
(active)
Draw a graph of the activity (see part (f)) as a function of time for a solution of zymogen
A after the addition of a small amount of protease A.
AA sidechains, 7.013 CG 2004, 3
at pH 7.0
STRUCTURES OF AMINO ACIDS
GENERIC AMINO ACID:
O
O
Individual amino acids
are linked through these
groups to form the
backbone of the protein.
O
O
H
NH3
+
O
H
NH3
+
O
H
N
+
C
H C CH2
NH3
+
O
N
C
H
O
O
C H
H
ISOLEUCINE
(ile)
HISTIDINE
(his)
O
O
O
C
S
CH3
H
NH3
+
METHIONINE
(met)
C CH2
C CH3
NH3
+
CH3
O
H
H
H
H C C CH3
C
C CH2
H
NH3
+
NH3 OH
+
TRYPTOPHAN
(trp)
H
O
O
C
H
C CH2
SERINE
(ser)
H
OH
NH3
+
H
H
O
O
C
C CH2
OH
NH3
+
C
H
H
H
THREONINE
(thr)
O
O
NH3+
LYSINE
(lys)
PROLINE
(pro)
H
C CH2CH2CH2CH2
NH3
+
C
H
C CH2
CH2
H
N
CH2
H +
H
N
C
H
O
O
H
O
C
H
PHENYLALANINE
(phe)
O
O
O
C H
GLYCINE
(gly)
O
C CH2
H
C H
NH3
+
LEUCINE
(leu)
H
NH3
+
H
NH2
C
C
C CH2CH2
C
O
O
O
C
O
C CH2CH2
O
ASPARTIC ACID
(asp)
GLUTAMINE
(glN)
NH3 CH3
+
H
NH3
+
O
NH3
+
H C C CH2CH3
H
NH2
C
H
GLUTAMIC ACID
(glu)
C
H
C
O
C CH2 C
O
O
C CH2CH2
H
ASPARAGINE
(asN)
O
NH3
+
CYSTEINE
(cys)
O
NH2
+
C
C CH2 SH
NH3
+
O
O
C
O
C
C
O
H C CH2 C
NH2
O
O
C
H
C CH2CH2CH2 N
ARGININE
(arg)
O
Peptide bonds
O
O
NH3
+
ALANINE
(ala)
H
Side chain, unique
to each differnt
amino acid
NH3
+
C
C CH3
O
H C R
O
C
H
O H R1
H
O H R
3
C
C
N
C
C
C
N
N
C
H
O H R2 H
Protein Synthesis
C
H
TYROSINE
(tyr)
H
CH3
C C
NH3 H
+
CH3
VALINE
(val)
S
S
GLY ALA VAL LEU ILE SER THR ASP ASN GLU GLN LYS ARG HIS TRP PHE TYR PRO CYS MET
G
A
V
L
I
S
T
D
N
E
Q
K
R
H W F
Y
P
C
M
Figure by MIT OCW.
AA sidechains, 7.013 CG 2004, 4