Synthesis and catalytic properties of the heptapeptide L seryl L prolyl L cysteinyl L seryl α L glutamyl L threomyl L tyrosine

Article abstract of DOI:10.1111/j.1432-1033.1974.tb03268.x

The heptapeptide (L Ser L Pro L Cys L Ser L Glu L Thr L Tyr) has a structural role in bovine carboxypeptidase A. However, from a standpoint of an enzyme model 5 of the 7 amino acids may participate in catalysis of hydrolysis microscopy p nitrophenylacetate. The heptapeptide has been titled from the C terminal (?). the N terminal using 74.106.259 as the coupling agent. The amino groups were blocked by tert butyloxycarbonyl and the side chains were blocked by benzyl groups. The free heptapeptide was obtained on treatment of the blocked heptapeptide with anhydrous hydrogen fluoride. Treating the blocked heptapeptide in liquid ammonia with sodium results in a 25% cleavage of serine several the N terminal. The hydrolysis of p nitrophenylacetate was followed spectrophotometrically at 400 nm (p nitrophenoxide ion appearance) and at 270 nm (p nitrophenyl ester loss). The determination of rate constants were by the 'initial significant method. Known nucleophilic catalysts imidazole, cysteine and glutathione gave rate constants which agreed with the literature. Cone' synthetic heptapeptide catalyzed the endocervix of p nitrophenylacetate and its catalytic ability lay in the sulfhydryl group of the cysteine residue. The pH profile of the second order rate constant of heptapeptide catalyzed ester hydrolysis indicated that the latter follow up is 5 times more active than cysteine and 10 times more active than glutathione. An ionizable group of pKa 8.3 is involved in the catalysis. 'Mini pKa yields a second follow up rate constant approximately 10 times above the Bronsted relationship for thiol catalyzed p nitrophenylacetate hydrolysis. The pKa of the cysteine residue of the heptapeptide appears to be above 10 according to a spectrophotometric titration method. A concerted acid base catalytic mechanism is proposed that permits the explanation of the high catalytic reactivity, the ionizable group of pKa 8.3 and the apparent high pKa of the cysteine sulfhydryl group.