Carboxypeptidase inhibitors binding

Fig. 34. Glu-72- Zn interactions in native carboxypeptidase A and in carboxypep-tidase A-inhibitor complexes (inhibitors have been reviewed by Christianson and Lipscomb, 1989). When substrates or inhibitors bind to the enzyme active site and interact with the zinc ion, the interaction of the metal with Glu-72 tends from bidentate toward uniden-tate coordination. The flexibility of protein-zinc coordination may be an important aspect of catalysis in this system, and the Glu-72->Zn - coordination stereochemistry observed here is consistent with the stereochemical analysis of carboxylate-zinc interactions from the Cambridge Structural Database (Carrell et al., 1988 see Fig. 4).

Interestingly, the successful design of captopril used simple chemical concepts guided by a hypothetical, paper-and-pencil model of substrate and inhibitor binding to the enzyme active site, that had been inferred from the crystal structure of bovine carboxypeptidase A. The X-ray structure of human ACE became available only in 2003, 25 years after the discovery of the captopril class of drugs. While the crystallographic analysis of the ACE complex... 

The crystal structure of the HNL isolated from S. bicolor (SbHNL) was determined in a complex with the inhibitor benzoic acid." The folding pattern of SbHNL is similar to that of wheat serine carboxypeptidase (CP-WII)" and alcohol dehydrogenase." A unique two-amino acid deletion in SbHNL, however, is forcing the putative active site residues away from the hydrolase binding site toward a small hydrophobic cleft, thereby defining a completely different active site architecture where the triad of a carboxypeptidase is missing. 

This analysis reveals that enzymes bind the transition state more tightly than the ground state by a factor approximately equal to the rate of acceleration (ie, Kjs/Ks kuncaJkcat)- This method has been used to show, for example, that the peptide phos-phonate inhibitors of carboxypeptidase A are true transition state analogs. 

In the case of carboxypeptidase B, Shaklai et al.(2lT> compared the relative contributions to the protein phosphorescence from tyrosine and tryptophan for the apoenzyme, the zinc-containing metalloenzyme in the absence of substrate, the metalloenzyme in the presence of the substrate iV-acetyl-L-arginine, and the metalloenzyme in the presence of the specific inhibitor L-arginine. The tyrosine tryptophan emission ratio of the metalloenzyme was about a factor of four smaller than that of the apoenzyme. Binding of either the substrate or the inhibitor led to an increase in the emission ratio to a value similar to that of the apoenzyme. The change in the tyrosine tryptophan phosphorescence ratio was attributed to an interaction between a tyrosine and the catalytically essential zinc. The emission ratio was also studied as a function of pH. The titration data are difficult to interpret, however, because a Tris buffer was used and the ionization of Tris is strongly temperature dependent. In general, the use of Tris buffers for phosphorescence studies should be avoided. 

In a recent study, Lin et al. (94a) has found that although the activity of PIR is low it is not zero. In carefully purified material assayed with C>p at pH 6 the Km was about twice that of RNase-A while the turnover number was 0.5% of the native value. This material bound the inhibitors 2 -CMP about 12 times less strongly than RNase-A. When Phe 120 is removed with carboxypeptidase from PIR to give des-(120-124)-RNase, all activity and binding properties are lost. 

Circumstantial evidence against the anhydride mechanism comes from the observation of Christianson and Lipscomb (1986) that the tight-binding inhibitor [110] (Kt = 0.2 pM) binds as the hydrate, rather than as an adduct with Glu-270, as would be expected were Glu-270 a nucleophile rather than an acid/base catalyst. In the complex of carboxypeptidase and the hydrate of... 

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