6.4 Carboxypeptidase A
Carboxypeptidase A is a protease that hydrolyses the C-terminal peptide bond in polypeptide chains. Whilst this enzyme demonstrates strict specificity with regard to the position of the amide bond (i.e. it must be C-terminal), it does not discriminate on the basis of the identity of the terminal residue; in fact, it will cleave off any residue with the exception of arginine (Arg), lysine (Lys) or proline (Pro). However, carboxypeptidase A is most efficient at removing terminal residues with aromatic or bulky aliphatic side-chains (Tyr, Trp, Phe, Leu, Ile).
The three-dimensional structure of carboxypeptidase A was elucidated by William Lipscomb at Harvard in 1967. It has a single polypeptide chain of 307 amino acid residues and an Mr of 34 500. It is a compact globular protein and is a metalloenzyme, having a tightly bound zinc ion that is essential for its activity. The zinc ion is in a pocket near the surface of the protein and is coordinated by a glutamate side-chain (Glu 72) and two histidine side-chains (His 69 and His 196). The substrate binds in the pocket, near to the zinc ion. Figure 44 shows a space-filling representation of carboxypeptidase A, with the zinc ion, Glu 72, His 69 and His 196 highlighted, and another representation, this time with a substrate bound. The substrate used to obtain this X-ray structure was glycyltyrosine (Figure 45), an analogue of the natural peptide substrate.
Carboxypeptidase A hydrolyses glycyltyrosine very slowly. Analogues of natural substrates, such as this molecule, that are processed very slowly by the enzyme, are often chosen for X-ray crystallographic studies of enzyme–substrate complexes. It is necessary to use substrate analogues because obtaining crystals for analysis can be a very lengthy process and the natural substrate would not remain bound to the enzyme for long enough to permit crystallisation. From the crystal structures of the enzyme–substrate complex, the mode of binding of glycyltyrosine to the enzyme has been deduced and is depicted in Figure 45. The tyrosine side-chain of the substrate occupies a non-polar pocket, whilst its terminal carboxyl group interacts electrostatically with the positively charged side-chain of arginine 145 (Arg 145). The NH hydrogen of the peptide bond is hydrogen-bonded to the OH group of tyrosine 248 (Tyr 248) and the carbonyl oxygen atom of the peptide bond is coordinated to the zinc ion. The terminal amino group of glycyltyrosine hydrogen-bonds, via an intervening water molecule, to the side-chain of Glu 270.
How do you think the binding of the substrate analogue, glycyltyrosine, to the active site of carboxypeptidase A would differ from that of a polypeptide substrate?
A polypeptide substrate would not have a terminal amino group in the active site, so would not form the interaction with Glu 270.
The interaction between Glu 270 and the glycyltyrosine molecule is thought to be responsible for the very slow rate of hydrolysis of this substrate analogue by the enzyme.
In binding substrate, the active site of carboxypeptidase A undergoes structural rearrangement to bring the groups that participate in catalysis into the correct orientation. This process is known as induced fit. The induced fit model of enzyme action was first proposed by Koshland. The side-chains of Arg 145 and Glu 270 both move 2 Å, water molecules are displaced from the non-polar pocket and, most striking of all, the phenolic hydroxyl group of Tyr 248 moves 12 Å when this residue swings into place for catalysis. This last movement is huge when you consider that, at its widest, the enzyme is only 50 Å across!
The movement of the hydroxyl group of Tyr 248 brings it from near the surface of the enzyme to the vicinity of the peptide bond to be hydrolysed. The different structural rearrangements effectively close the active-site pocket, excluding water and making the environment of the active site hydrophobic. Clearly, a peptide substrate could not access the active site if it was in this closed conformation. To permit substrate binding, carboxypeptidase A has to have a very different conformation in the unbound state than it does in the catalytically active state. Thus in undergoing these substantial rearrangements, induced by the substrate, it creates the correct environment for catalysis.
The mechanism of catalysis of the peptide bond by carboxypeptidase A is illustrated in Figure 46. The carbonyl group of the peptide bond is coordinated to the zinc ion, making the C=O bond more polarised than usual. This effect is enhanced by the non-polar environment of the zinc ion, which increases its effective charge. In this way, the zinc ion stabilises the negative charge that develops on the O atom (electrophilic catalysis). This large dipole makes the C atom of the carbonyl group more vulnerable to nucleophilic attack, because it has a partial positive charge. The negatively charged Glu 270 removes a proton from a water molecule and the resulting OH– directly attacks the vulnerable carbonyl C atom (general base catalysis). Tyr 248 simultaneously donates a proton to the NH group of the peptide bond. In this way, the peptide bond is hydrolysed.
Carboxypeptidase B specifically removes C-terminal Arg or Lys residues from peptides. Whilst being very similar to carboxypeptidase A in terms of overall structure, carboxypeptidase B has a negatively charged aspartate (Asp) side-chain in an appropriate position to bind the positively charged side-chains of Arg and Lys.