Glycyl-L-tyrosine

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. 

The determination of the crystal structure of carboxypeptidase A and its complex with glycyl-L-tyrosine has been completed to 2.0 A resolution by Lipscomb and coworkes (91). A detailed description of the molecule including a comprehensive discussion of structure-function relations have been presented (91). 

The binding of glycyl-L-tyrosine in the active site pocket of carboxypeptidase A is illustrated in Fig. 15. Tyrosine-248 and glutamic acid-270 are believed to participate in the catalytic reaction and represent the acidic and basic groups, respectively, involved in the bell-shaped pK-rate profile. In the bond-cleavage reaction, the carboxyl group of Glu-270 may act by a nucleophilic attack on the carbonyl group while Tyr-248... 

Fig. 15. Schematic drawing of the active site region of carboxypeptidase A interacting with glycyl-L-tyrosine. From Lipscomb et al. (91) and sequence information from the laboratory of H. Neurath (101).

It is difficult to make water-soluble peptides form complexes with titanium alkoxide, because they are not soluble in organic solvents. Therefore, such templates cannot be imprinted by the complexation approach. Instead, they could be imprinted in Ti02-gel films by the alternate adsorption approach with Ti(0 Bu)4. Figure 6.24a shows a plot of alternate layer-by-layer assembly of 100 mM titanium butoxide (3 min adsorption in toluene/ethanol) and 10 mM glycyl-L-tyrosine (Gly-L-Tyr, 10 min adsorption in water). The template molecule was removed by treatment with 10 mM aqueous sodium hydroxide, as... 

GLTLYR10. Glycyl-L-tyrosine dihydrate [CuH14N204, 2(H20)]. Cotrait M, Bideau J-P (1974) Acta Crystallogr, Sect B 30 1024... 

Fig. 12. Difference spectra of the individual aromatic amino acid residues (Pig. 12a) and of three proteins (Fig. 12b) solution of pH 6 to 7 in sample beam and solution of pH 1 to 1.5 in reference beam. Curve 1 10 M glycyl-n-phenylalanine curve 2.5 X 10 M glycyl-L-tyrosine and curve S 5 X 10 M glycyl-L-tryptophan. (Yanari and Bovey, 1960.)...

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). 

D. W. Smits and E. H. Wicbenga. Acta Cryst. 6, 531-9 (1953). x-ray crystal structure of glycyl-L-tyrosine hydrochloride. 

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.

Mixed Polypeptides. Glycyl-alanine anhydride glycyl-l-tyrosine anhydride glycyl-I-phenylalanine anhydride... 

Optically Active. Glycyl-l-tyrosine. Glycyl-d-alanine. Glycyl-d-tryptophane. Glycyl-l-phenylalanine. Glycyl-3, 5-diiodo-l-tyrosine. dl-Alanyl-d-alanine. 1-AlanyI-glycine. d-Alanyl-d-alanine. d-AIanyl-l-leucine. d-Alanyl-d-tryptophane. 

The synthesis of polypeptides composed of different amino acids is most easily effected by this method. Those containing tyrosine are of particular interest, since the first natural tetrapeptide was isolated from silk in 1907, and was composed of glycine, alanine and tyrosine. Twelve isomers are possible for a tetrapeptide, of this composition, but if the results of partial hydrolysis and subsequent anhydride formation be taken into account, this number is reduced to eight. Of these, glycyl-d-alanyl-glycyl-l-tyrosine was synthesised in 1908 by combining chloracetyl-d-alanyl-glycylchloride with 1-tyrosine ester —... 

An attempt was made at the same time to prepare the isomeric d-alanyl-glycyl-glycyl-l-tyrosine it failed on account of the difficulty of preparing pure a-bromopropionyl-glycyl-glycyl-chloride, but there seems no reason to suppose that Fischer will not overcome this small difficulty in preparing a desired compound, when he has overcome such vast difficulties already in connection with the synthesis of the polypeptides. 

Glycyl-d-alanyl-glycyl-l-tyrosine (chloracetyl-d-alanyl-glycyl chloride + 1-tyrosine ester). 

In general, the solubility in water of mixed polypeptides is greater than the solubility of the polypeptides made up of a single amino acid the ready solubility of the dipeptides glycyl-l-tyrosine, leucyl-tyrosine, which contain the amino acids soluble with difficulty in water, should also be noted. 

Glycyl-l-tyrosine is readily hydrolysed by trypsin, but not by pepsin, and it therefore serves an excellent compound for determining whether a given proteolytic ferment behaves as a peptic or a tryptic enzyme. For this reason it was employed by Abderhalden and Rona to determine the nature of the enzymes contained in the pyloric and duodenal... 

Abderhalden and Teruuchi used glycyl-l-tyrosine to determine the nature of the enzymes in yeast juice, i.e. endotryptase, in papain and in the juice of nepenthes. The two former hydrolysed it, and consequently they contain tryptic enzymes the last had no action upon it, and the enzyme of nepenthes is therefore like pepsin in its action. These results confirm the observations of other investigators, and the confusion concerning the nature of these enzymes would appear to be now settled with certainty. 

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. 

At the same time another diketopiperazine, glycyl-l-tyrosine anhydride, was also obtained its nature was established a little later by identification with synthetical glycyl-l-tyrosine anhydride prepared from the ester of chloracetyl tyrosine and ammonia. In one experiment its yield amounted to 4 2 per cent, of the silk-fibroin employed. 

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