Glycyl-tyrosine substrate

Figure 7-3. Two-dimensional representation of a dipeptide substrate, glycyl-tyrosine, bound within the active site of carboxypeptidase A.

We chose carboxypeptidase A (CPA) as the protein to study first because relatively high accuracy X-ray data was available both for the native protein [6] and for the protein with the substrate, glycyl-tyrosine, (gly-tyr) bound to it [7]. 

Fig. 11. The slowly hydrolyzed substrate glycyl-L-tyrosine binds to carboxypeptidase A in a nonproductive complex where the amino-terminal glycine complexes the active-site ion (large sphere) to form a five-membered chelate, as in Fig. 10. Protein-bound zinc ligands Glu-72, His-69, and His-196 complete the coordinadon polyhedron of pentacoordinate zinc. Active-site residues are indicated by one-letter abbreviadons and sequence numbers E, glutamate H, hisddine R, arginine Y, tyrosine. [Reprinted with permission from Christianson, D. W., Lipscomb, W. N. (1986) Proc. Natl. Acad. Sci. U.S.A. 83,7568-7572.]...

The tyrosine group can be coupled with p-azobenzenearsonate to give an asymmetric centre. The CD spectrum changes on addition of glycyl-L-tyrosine, showing that the conformation of the azotyrosyl residue is altered on binding of substrate. 

Another difficulty which has faced the x-ray crystallographic approach to deciphering enzyme mechanisms is the inability to examine enzyme-substrate complexes. To circumvent this problem, complexes with inhibitors, products, or pseudosubstrates have been employed. In the case of carboxypeptidase A, mechanistic deductions have been based on results obtained using the very poor substrate glycyl-L-tyrosine (60). 

The model was built and optimized in the absence of inhibitors. Dipeptide inhibitors were docked in the eneigy-minimized model. The coordinates of the slowly hydrolyzed substrate glycyl-L-tyrosine [114] for CPA were used to guide the (manual) docking. Atomic coordinates were obtained from the Protein Data Bank [115], Energy minimization and molecular dynamics were carried out with GROMOS [116]. 

Figure 6 Amino acids in the active site of carboxypeptidase N. The dipeptide glycyl-L-tyrosine is docked into the active site. The active site zinc is coordinated to His 69, Glu 72, and His 196. Arg 145, Asn 144, and "tyr 248 provide specificity for substrates bearing a free terminal carboxylate. Gin 255 forms a hydrogen bond with the side chain of the carboxy-terminal arginine or lysine. Glu 270 promotes the nucleophilic attack of a water molecule at the scissile peptide bond.

Figure 1-6. The structure of carboxypeptidase A changes dynamically upon substrate binding. (A) Enzyme alone, (B) enzyme complex with glycyl-tyro-sine. Tyrosine 248 moves 12 A after binding of substrate. Hydrolysis results as a concerted action of Zn2+, Clu, Tyr, and Arg side chains towards the carbonyl and nitrogen group in the susceptible peptide bond (C).

On account of these results with serum Abderhalden and Rona investigated the action of human blood serum on glycyl-l-tyrosine in certain cases of disease, as also the urine. In some diseases no hydrolysis occurred, but in other diseases there was distinct hydrolysis. As yet no conclusions can be drawn from these results, as they require amplification both as regards the enzyme solution and the substrate. In no case had the urine any action upon glycyl-l-tyrosine this seems at variance with the presence of an urotryptic enzyme which Cathcart studied in its action upon proteins. 

Trypsin, chymotrypsin, and cathepsin C (all from bovine), and the synthetic substrates glycyl-L-phenylalanine p-naphtylamide (GPNA), 4-phenylazo benzyloxy-car-bonyl-pro-leu-gly-pro-D-arg (PZ-peptide), N-benzoyl-L-tyrosine ethyl ester (BTEE), and Na-benzoyl-DL-arginine p-nitroanilide (BAPNA), were purchased from Sigma Chemical Co (St. Louis, MO). Fresh bluefish (Pomatomus saltatrix) and sheephead samples were purchased from a local fish market (Waldman Plus, Montreal, PQ) and kept in iCe until ready for use. 

---