Chymotrypsin Is Activated by Proteolysis Adapted from Campbell (1999) Biochemistry (3d) p.179 245 R15-I16 Chymotrypsinogen (inactive) p -Chymotrypsin (active)

Chymotrypsin Is Activated by Proteolysis

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245

R15-I16

Chymotrypsinogen (inactive)

-Chymotrypsin (active)

S14-R15 T147-N148

Trypsin

-Chymotrypsinogen (active)

-Chymotrypsin

I16L13 A149Y146

Disulfide bonds

Ch

arg

e R

ela

y in

Active S

ite

Ser195

His 57

Asp 102

H–O–CH2

O

C–O -

=

Active Ser

H–N N

C C

C

H

H

CH2

Ser195

His 57

Asp 102

- O–CH2

OC–O–H

=

N N–H

C C

C

H

H

CH2

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pH Influences Chymotrypsin Activity

5 6 7 8 9 10 11

pH

Relative

Activity

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162

pH Influences N

et Charge of P

rotein Jua

ng

RH

(2

00

4)

BC

ba

sics

+Net Charge of a Protein

Buffer pH

Isoelectric point,pI

-

3

4

5

6

7

8

9

10

0+

Imidazole on Histidine Is Affected by pH

H–N N

C C

C

H

H

H+

pH 6 pH 7

+H–N N–H

C C

C

H

H

Inactive+ Ser

195

His 57

Asp 102

H–O–CH2

O

C–O -

=

H–N N–H

C C-H

C

CH2

H

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158

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Chymotrypsin Produces New Ile16 N-Terminal

I16L13 Y146

Asp 194

–CH2COO-

Ile 16NH2–

Ile 16+ NH3–

5 6 7 8 9 10 11pH

Relative activity

pH 9 pH 10pKa

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165

New NH2-terminus

New Ile16 N-Terminal Stabilizes Asp194

Asp 102

His 57 Ser 195

Asp 194

Gly 193

Ile 16

+NH3

Catalytic Triad

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.206

Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.112

O (CH3)2CH–O– P –O–CH(CH3)2

F

=

Chymotrypsin Ser195 Inhibited by DIFP

Diisopropyl-fluorophosphate (DIFP)

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

O-…H

CH2

Ser 195

O (CH3)2CH–O– P –O–CH(CH3)2

=

O

CH2

Ser 195

XXXX

Addition of Substrate Blocks DIFP Inhibition

Reaction time

Percent Inhibition of activity (%

)

100

50

0

No substrate

Add substrate

S

+ DIFP

+ DIFP & substrate

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

XXXX

Chymotrypsin Also Catalyzes Acetate

O-C N- H

O-C O-

Peptide bond

Ester bond

O

CH3–C–O– –NO2

Nitrophenol acetate

HO– –NO2

O

CH3–C–OH

Hartley & Kilby

Chymotrypsin+ H2O

Nitrophenol

Acetate

No acetate was detected at early stage

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168

O -

C

Time (sec)N

itrop

hen

ol

Two-Stage Catalysis of Chymotrypsin

O

CH3–C–O– –NO2

Nitrophenol acetate

OC

O

CH3–C HO– –NO2

+ H2O

O-HC

CH3COOH

Kinetics of reaction

Two-phasereaction

Acylation

Deacylation (slow step)

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169

Extra Negative Charge Was Neutralized

O-C N- H

O-C-OH

NH2-

-C-C-N-C-C-N-C-C-N- H H

E + S

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179

O -

-C N- HO H

O -

-C N- HO H

Active Site Stabilizes Transition State

Asp 102

His 57

Met 192

Gly 193

Asp 194Ser 195

Cys 191

Catalytic Triad

Thr 219

Ser 218Gly 216

Ser 217

Trp 215

Ser 214

Cys 220

Specificity Site

Active Site

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