Download 7.014 Section Problem:

7.014 Section Problem:
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 amino acid(s).
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 G are any amino acid and X is one of the specific amino acids uniquely
recognized by the enzyme. If this process repeats over and over, the substrate will be completely
degraded into small peptide fragments.
Each member of the family cleaves after a specific amino acid of the substrate because that amino
acid is recognized by a binding pocket of the enzyme that is specifically designed to bind that
particular amino acid.
a) One of these protease enzymes, trypsin, cleaves after lys or arg. What would be the product(s)
if each of the following 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 are shown below (not to scale – the actual enzyme
is much larger than the binding pocket). If the amino acid side chain of the substrate binds well to
the “recognition pocket” the substrate will bind and be 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 specificities 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
c) How could 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. You then take identical small samples from this
solution at regular intervals over the next few hours as the solution stands at room temperature
and measure their activity (the rate at which they cleave the substrate protein). You find that the
activity of the enzyme drops rapidly as time passes.
However, if you add a large excess of casein (a protein found in milk which has no
enzymatic activity), the protease loses its activity much more slowly. These data are sketched
below:
activ ity
100
protease A + casein
protease A alone
0
0
tim e
Explain these observations.
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 once – the 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?
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
H
C
CH3
H
O
CH2 SH
H
NH3
+
H
N
C
O
+
C
CH2
NH3
+
N
C
H
H
H
O
C
CH2CH2
S
CH3
H
NH3
+
C
O
CH2CH3
H
C
H
CH2
H
CH3
NH3 OH
+
THREONINE
(thr)
C
CH2
H
H
H
NH3
+
H
H
O
CH2CH2CH2CH2
LYSINE
(lys)
H
O
O
C
H
C
C
H
O
O
C
CH2
OH
NH3
+
H
TYROSINE
(tyr)
H
OH
SERINE
(ser)
C
H
CH2
NH3
+
PROLINE
(pro)
O
C
NH3
+
C
H C CH2
CH2
H
N
CH
2
H +
H
H
H
CH3
O
O
H
C
TRYPTOPHAN
(trp)
C
CH3
H
O
C
LEUCINE
(leu)
N
C
GLYCINE
(gly)
O
NH3
+
H
C H
NH3
+
H
PHENYLALANINE
(phe)
O
H
NH2
C
CH2
NH3
+
METHIONINE
(met)
O
O
C H
C
C
O
O
O
O
CH2CH2
C
C
C
C
C
GLUTAMINE
(glN)
O
C H
O
O
NH3
+
O
ASPARTIC ACID
(asp)
O
C
CH2 C
NH3
+
O
H
O
ISOLEUCINE
(ile)
C
H
C
O
C
NH2
C
NH3 CH3
+
H
HISTIDINE
(his)
H
CH2CH2
H
ASPARAGINE
(asN)
GLUTAMIC ACID
(glu)
CYSTEINE
(cys)
C
NH3
+
O
C
CH2 C
O
NH3
+
O
NH2
C
O
C
NH2
+
C
C
O
C
O
O
C
H
CH2CH2CH2 N
H
O
O
C
H
C
Peptide bonds
O
O
ARGININE
(arg)
O
O
Side chain, unique
to each differnt
amino acid
NH3
+
ALANINE
(ala)
H
C R
NH3
+
C
NH3
+
O
C
O
C
O H R1 H
O H R
3
C
C
N
C
C
C
N
N
C
R
H
2
H
H
O
Protein Synthesis
H
C
CH3
C
NH3 H
+
CH3
VALINE
(val)
NH3+
Solutions to: Enzymes/Protein Structure
a)
i) leu-thr-phe-ala-ser (unchanged)
GENERIC ENZYME:
ii) trp-tyr-lys + ala-phe
iii) 2 lys + 2 arg
Schematics of enzymes: (side chains shown in black)
ACTIVE SITE peptide bond cleavage
gly
gly
gly
gly
ile
-
asp
gly
protease A
val
phe
protease C
protease B
RECOGNITION POCKET specific substrate regognition
Schematics of substrates:
lysine, arginine
peptide backbone
phenylalanine,
tryptophan, tyrosine
glycine, alanine
+
long & + charged
small
large & hydrophobic
b) Matching them up:
protease A - large open pocket. Could be lys/arg or phe/trp/tyr.
protease B - large open pocket with (-) charge at bottom. Therefore, lys/arg, which
means that protease A must cut after phe/trp/tyr
protease C - small pocket. Cuts after Gly, ala.
c) Change the asp in the bottom of the pocket in protease B to a lys or arg.
d) It might still bind lys or arg, but if the space in the pocket were constrained, there might not be
enough room because glu is longer than asp.
e) The others are required to hold the essential ones in place.
f) Protease A is a protein, therefore other protease A molecules can cleave it and thereby
inactivate it. Having casein around decreases the chance that a protease A molecule will cleave a
protease A molecule, because it will be more likely to cleave the more numerous casein
molecules.
g) The protease enzyme must be able to bind to the target amino acids. If they are buried inside
the target protein, the protease can't "see" them and therefore can't cut at them. Eventually, the
structure of the target protein gets so broken down that the inside amino acids are exposed to the
protease.
h) To protect protease A from degradation by other protease A molecules, bury all the
phe/trp/tyr inside the protein so that they cannot be recognized and cleaved.