Carboxypeptidase A

• Digestive Enzyme: Zn Peptidase Family

• Exopeptidase, cuts from C-terminal
• Prefers bulky & aromatic side chains
• 309 aa’s : MW = 34,760 : pI ~ 6.1
 (polypeptide)n + H2O

Carboxypeptidase A (CPA)

(polypeptide)n-1 + C-terminal
amino acid
aromatic preferred
 ARTIFICIAL SUBSTRATE
Gly-Tyr

H H
| |
H3N+ - C - C - N-CH-CH2- -OH
| || |
H O COO-
 Active site

• Coordinated Zn 2+ bound to His-69, Glu-72, His-

196 & H2O
• A grove for polypeptide substrate binding
• Hydrophobic pocket for binding the side chain of
the C-terminal amino acid
• Terminal -COOH gp forms es interaction with
Arg-145
 Y248-OH
HOH
HOH
HOH
HOH
HOH
HOH HOH
E270
Zn +R127 +R145
H196 E72
 Substrate binding induces
conformational change
• Induces movement of Arg-145, Glu-270 &
Try-248 to new positions close to substrate
forcing water molecules out of the active
site & creating a hydrophobic environment
 TYR

GLU
ARG
 TYR

GLU
ARG
bonds contributing
Y248
to KM & binding
step. OH
H H
- CH-C-N-CH-CH2- O -OH
E270 O COO-
Zn +R127 +R145
H196 E72
 Y248Y248
OH OH
H H
-CH-C-N-CH-CH2- O -OH
H-OH O COO -
E270
Zn +R127 +R145
H196 E72
 Mechanism

• Carbonyl O of the peptide bond to be hydrolysed

replaces the water molecule on Zn 2+
• Metal facilitates cleavage by withdrawing the e-
from the carbonyl gp
• Glu-270 acts as general base & withdraws a H+
from water to create a nucleophile
• Tyr-248 donate H+ to the N of peptide bond being
hydrolysed
 Y248
OH
H H
-CH-C-N-CH-CH2- O -OH
H-OH O COO -
E270
Zn +R127 +R145
H196 E72
 Concerted Mechanism of Catalysis
Carboxypeptidase A

Active (248)
(270)
site Tyr
Glu 3 4 ACTIVE
pocket O - Site for
COO - H SITE specificity
H
+
H O-
C N R 5
N C C
2
Substrate O-

Juang RH (2004) BCbasics

peptide
chain
1 + COO - +
Arg (145)
His
(196)
Zn Glu C-terminus
(72) Check for
His (69)
C-terminal
 Ribonuclease A
• Bovine pancreatic RNAse A - first enz to have
complete amino acid sequence determined
(Smyth, Stein & Moore, 1963)
• Catalyses cleavage of phosphodiester bond of
RNA
• Acid-base catalysis
• Reaction involves transfer of P from 5' position of
one nucleotide to 3' position of the next nucleotide
in the chain
 RNase A : Acid-base catalysis
• His 12 NH2

– (general base) N
N

– abstracts a
proton from O
N
N NH
His 12
2’ hydroxyl of -O P O
H
CH O
O
3’ nucleotide O- H H
N CH2
C

• His 119 H H
NH2
O H N
– (general acid) O
N

– donates a -O P O
O
proton to 5’ O-
H
H H
N O

hydroxyl of His 119 N+

C H H
nucleoside O H2 OH OH
C
CH N
H
HN
 NH2

RNase A N
N

cyclic intermediate N
O N NH
• Net proton His 12
-O P O CH
transfer O H
N CH2
C
O

H H
from O-
NH2
His119 to H
O
H
H N
His 12 O
N

-O P HO
O
N O
O- H H
His 119 N
C H H
O H2 OH OH
C
CH N
H
HN
 NH2

Rnase A N

Product release
N

N
O N NH
• Water His 12
-O P O CH O
replaces the O H
N CH2
C
H H
released O-

nucleoside H
O
H
H N

• Acid and O

base roles -O P O H
are reversed
O- H
for H12 and His 119 N
H119 O
C
H2
C
CH N
H
HN
 NH2

RNase A
N
N

N
O N NH
• Original His 12
-O P O CH O
Histidine O H
N CH2
C
H H
protonation O-

states are H
OH
H
N
restored O

-O P O H

O- H
His 119 N
C
O H2
C
CH N
H
HN