Review Session: Monday, Dec' 8

1
Review Session Monday, Dec. 8 6 pm BI
212 Exam Wednesday, Dec. 10 1030 am FH 4
2
Holiday Concerts Whatcom Chorale Sunday, Dec.
14 3pm and 730 pm WWU PAC Concert Hall Music
by Charpentier, Saint-Saens, Berlioz and some
French carols.
3
Figure 14-15 Effect of pH on the initial rate of
the reaction catalyzed by the enzyme fumarase.
Page 487
4
Catalytic Mechanism Determination
1) kinetic analysis - what is the kinetic
signature? - mode of inhibition revealed
- determine rates for individual steps - does
order of addition matter? (sequential vs.
Ping-Pong) 2) active site modification
(irreversible inhibitors) - derivatize protein
identify the modified sidechain(s) 3) structure
determination (e.g. RNase A, lysozyme, serine
proteases)
5
Figure 15-3 The bovine pancreatic RNase
Acatalyzed hydrolysis of RNA is a two-step
process with the intermediate formation of a
2,3 -cyclic nucleotide.
Reverse rxn with water as leaving group
His 12 general base Nucleophillic attack His 9
protonates leaving group
Page 499
general acid/base catalysis
6
Figure 15-2 The pH dependence of Vmax/KM in the
RNase Acatalyzed hydrolysis of cytidine-2,3
-cyclic phosphate.
Page 499
7
Enzymatic catalysis proceeds by one or more of
1) general acid/base catalysis (GABC) 2)
covalent catalysis 3) Metal ion catalysis 4)
electrostatic stabilization 5) proximity effects
6) preferential stabilization of the
8
Covalent catalysis
Is characterized by the formation of a covalent
Enz-S adduct that alters the reaction
pathway Nucleophiles many amino acid side
chains (H, K, C, S, D, E, Y), some coenzymes
(TPP) Electrophiles some coenzymes (e.g. PLP)
9
Stryer Fig. 9.1
10
Stryer Fig. 9.2 Identifying an active ser--Out
of 28 ser, only 195 is labeled
11
Figure 15-19 Reaction of TPCK with chymotrypsin
to alkylate His 57.
Page 517
An affinity label
12
This assay allows the use of ester hydrolysis to
help formulate a kinetic model for catalysis by
chymotrypsin.
Stryer Fig. 9.3 Chromogenic substrate (like V2
p. 516)
13
Slow hydrolysis of ES
Fast release of PNP
Stryer Fig. 9.3 Kinetics indicate a 2-step
reaction
14
Figure 15-18 Time course of p-nitrophenylacetate
hydrolysis as catalyzed by two different
concentrations of chymotrypsin.
Page 516
15
Figure 15-20a X-Ray structure of bovine
trypsin.(a) A drawing of the enzyme in complex
with substrate analog.
Page 518
Similar backbone structrures for chymotrypsin and
elastase.
16
Figure 15-20b X-Ray structure of bovine trypsin.
(b) A ribbon diagram of trypsin.
Page 519
17
Figure 15-21 The active site residues of
chymotrypsin.
Page 520
Catalytic Triad
18
Stryer Fig. 9.5 Covalent catalysis
19
Figure 15-25a Transition state stabilization in
the serine proteases. (a) The Michaelis complex.
Page 524
20
Figure 15-25b Transition state stabilization in
the serine proteases. (b) The tetrahedral
intermediate.
Page 524
Preferential binding to transition state 3 new
H bonds from after distortion
21
Figure 15-23 Catalytic mechanism ofthe serine
proteases.
His 57 GB Stablilized by H bond to asp 102
Page 522
Reverse His 57 GA
22
Table 15-4 A Selection of Serine Proteases.
Page 516
23
Specificity of ser proteases determined by
different binding pockets Chy--slit lined by
hydrophobic residues Tryp- asp at bottom of
pocket Elastase Pocket blocked by val-thr
midway down the pocket.
24
Figure 15-20cX-Ray structure of bovine trypsin.
(c) A drawing showing the surface of trypsin
(blue) superimposed on its polypeptide backbone
(purple).
Page 519
25
(No Transcript)
26
Example of convergent evolution.
27
Figure 15-22 Relative positions of the active
site residues in subtilisin, chymotrypsin, serine
carboxypeptidase II, and ClpP protease.
From E. coli
From wheat germ
Page 521
28
Figure 15-27 Activation of trypsinogen to form
trypsin.
Page 527
29
Figure 15-28 Activation of chymotrypsinogen by
proteolytic cleavage.
Page 528
30
Figure 15-24a TrypsinBPTI complex. (a) The
X-ray structure shown as a cutaway surface
drawing indicating how trypsin (red) binds BPTI
(green).
Page 523
31
Figure 15-24b TrypsinBPTI complex. (b) Trypsin
Ser 195, the active Ser, is in closer-than-van
der Waals contact with the carbonyl carbon of
BPTIs scissile peptide.
Page 523