ENZYMES Proteins  Elmhurst College
Lock and Key Theory Coenzyme - NAD+ Enzyme Regulation  Chemistry Department
Carboxypeptidase Enzyme Inhibitors  Virtual ChemBook

Carboxypeptidase

Introduction:

Carboxypeptidase is an enzyme synthesized in the pancreas and secreted into the small intestine. This enzyme hydrolyzes the first peptide or amide bond at the carboxyl or C-terminal end of proteins and peptides. It has a stronger preference for those amino acids that have aromatic or branched hydrocarbon chains.

In the graphics and chime, the substrate is the dipeptide - tyrosine-glycine - usually shown as magenta or space filled atom colors.

Most General: Carboxypeptidase-1 - Chime in new window

General Active Site: Carboxypeptidase-2 - Chime in new window

Detailed Active Site: Carboxypeptidase-3 - Chime in new window

Metal Ion Cofactor:

The enzyme contains zinc (II) ions as a metal ion cofactor which is at the heart of the active site.

The zinc ion is coordinated in a tetrahedral geometry first by three amino acid side chains in the active site: through Nitrogen from histidine-69, through N from his-196, and through Oxygen from glutamic acid-72. The fourth bonding position is either a water molecule or an oxygen from the carbonyl of glycine-1 as shown in the graphic on the left.
(Color Key: Red = O; blue = N; Cyan = Zn; gray = C; magenta = substrate- peptide backbone; thin white,red,blue line = backbone of rest of protein in enzyme)


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Substrate in the Active Site:

The active site provides nearest neighbor amino acid side chains that interact with the substrate. There are several regions of interest.

1) There is a hydrophobic pocket which is not shown that accommodates the aromatic and branched hydrocarbon side chains on the the substrate peptide.

2) Arginine-145 plays a major role in salt bridge binding to the carboxyl group of the tyrosine in the dipeptide - gly-tyr. Arginine has two terminal NH3+ groups that bind ionically with the negatively charged oxygen of the carboxyl group.

3) Asparagine-144 is also in the vicinity and may play a hydrogen bonding role.

4) Tyrosine-246 appears to move a distance of up to 12 Angstroms, in an example of an induced fit, upon binding of the substrate. It was once thought that it may hydrogen bond to the N of the amide to be hydrolyzed. Further experiments where phenylalanine replaced tyr-246 showed no effect on the rate of catalytic action. Therefore, tyr-246 now appears to also perhaps hydrogen bond to the substrate carboxyl group.

Most General: Carboxypeptidase-1 - Chime in new window

General Active Site: Carboxypeptidase-2 - Chime in new window

Detailed Active Site: Carboxypeptidase-3 - Chime in new window

 
Click for larger image 

Mechanism of Reaction at the
Active Site with Substrate Interaction:


The objective of the carboxypeptidase enzyme is to hydrolyze peptides at the amide bond on the C-terminal end of the chain. In the example the dipeptide is gly-tyr. By definition the tyr is on the C-terminal end. The amide bond is between the gly and the tyr.

Various mechanisms have been proposed for how this bond is broken as shown by the red jagged line in the graphic on the left.

It was once thought that the carbonyl bond is activated by interaction with the Zn ions. This leads to the addition of -OH from water to the carbonyl to produce an acid and the ultimate rupture of the C-N bond.

The current proposed mechanism is that arg-127 interacts with the carbonyl as shown on the far left. The zinc ion still maintains a bond with water which is further activated by glu-270. It is then the hydroxide attack on the carbonyl carbon which results in the breaking of the amide bond.

The major idea that should be derived from the details of this example of an enzyme controlled reaction is that various changes in the electronic environment of the substrate caused by the close proximity of various side chains in the enzyme cause a more rapid reaction to occur.

Most General: Carboxypeptidase-1 - Chime in new window

General Active Site: Carboxypeptidase-2 - Chime in new window

Detailed Active Site: Carboxypeptidase-3 - Chime in new window