Synthesis and catalytic properties of the heptapeptide L-seryl-L-prolyl-L-cysteinyl-L-steryl-alpha-L-glutamyl-L-threonyl-L-tyrosine.

Abstract

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 five of the seven amino acids may participate in catalysis of hydrolysis of p‐nitrophenylacetate. The heptapeptide has been synthesized from the C‐terminal to the N‐terminal using dicyclohexylcarbodiimide 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 from 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 slope” method. Known nucleophilic catalysts imidazole, cysteine and glutathione, gave rate constants which agreed with the literature. The synthetic heptapeptide catalyzed the hydrolysis 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 molecule 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. This pKa yields a second‐order rate constant approximately 10 times above the Brosted 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.