Carboxypeptidase flexibility

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).

Figure 7 " Cd PAC spectra for carboxypeptidase A under different physical conditions. Fourier transform of the experimental data (dotted hne) and Fourier transform of the fit (hold line). The fine hroadening foimd in solution as compared to the crystalline state indicates that the metal ion hinding site is strucmrahy more flexible in solution. (Reprinted with permission from Bauer, Danielsen, Hemmingsen, Sorensen, Ulstrup, Friis, Auld and Bjerrum. 1997 American Chemical Society)...

Bromoacetyl-p-aminobenzyl succinic acid alkylates a methionyl residue in carboxypeptidase B. As shown above, similar reagents modify amino acids other than methionyl residues, i.e., Tyr-248 and Glu-270. In those cases, the alkylating moiety was located in close proximity to the scissile bond of a normal substrate. Therefore, it would most likely interact with a residue that presumably functions in the catalytic step. The hydrophobic nature of bromoacetyl-p-aminobenzylsuccinate and the fact that the reactive side chain is carried on the aromatic part of the molecule suggests that the methionyl residue modified is part of the substrate recognition site in the hydrophobic pocket of carboxypeptidase B.i.0,16 This possibility is reminiscent of the role of Met-192 in chymo-trypsin, where it appears to function as a flexible hydrophobic lid on the substrate binding pocket. 

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