Carboxypeptidase chemical modification

Potts et al. 89) have shown that the 5 C-terminal residues of S-peptidfe can be removed with carboxypeptidase. The resulting derivative (residues 1-15) forms a strong complex with S-protein having full catalytic activity. It is clear from the X-ray structure that these 5 residues interact little, if at all, with any part of S-protein, and they are remote from the active site. The various changes produced in this component by synthesis and by chemical modifications are discussed later. 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

The ionizable ligand, EH2, is assigned to Glu270, since chemical modification of this residue with CMC (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide) decreases the binding affinity of carboxypeptidase A for zinc and lead by more than 60- and 200-fold, respectively. A bridging interaction between the Glu270-coordinated zinc hydroxide is implicated by the ability of Zn and Pb to induce a marked increase in the visible absorption spectrum of the cobalt enzyme within 20 ms of mixing 10 " M Zn or Pb with 10 " M cobalt enzyme. 

Carboxypeptidase A is one of the most intensely investigated zinc metalloenzymes. The enzyme as isolated contains 1 g-atom of zinc per protein molecular weight of 34,600. Removal of the metal atom either by dialysis at low pH or by treatment with chelating agents gives a totally inactive apoenzyme (46). Activity can be restored by readdition of zinc or one of a number of other di-valent metal ions (47). Through a combined use of chemical modification and transient state kinetic studies, it has been possible to determine the role of zinc in the catalysis of ester and peptide hydrolysis by this enzyme. 

The substitution of another metal for that present in the native state or the removal of any metal is the simplest chemical modification for a metalloenzyme. Marked changes in activity are usually observed in either case. Substitution of cadmium for zinc first demonstrated a difference in the esterase and peptidase activities of carboxypeptidase A (47). The activity of [(CPD)Cd] toward Bz-Gly-L-OPhe is increased, but that enzyme is virtually inactive toward Cbz-Gly-L-Phe. 

---