The reactivity of thiol-subtilisin, an enzyme containing a synthetic functional group

L. Polgár, Myron L. Bender

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Abstract

A synthetic enzyme was prepared by transforming the reactive serine residue in the active site of subtilisin to a cysteine residue. The transformation was accomplished nearly quantitatively by treating the serine residue with phenylmethanesulfonyl fluoride (PMSF), displacing the PMS group with thiolacetate ion, and allowing the acetyl-thiol-subtilisin to deacylate enzymatically. Titration with p-mercuribenzoate (PMB) and amino acid analysis indicated the presence of thiol groups and cysteine residues, respectively. Thiol-subtilisin catalyzes the hydrolysis of p-nitrophenyl acetate. This reaction, which is inhibited by p-mercuribenzoate, is characterized by a presteady-state burst of p-nitrophenol followed by a zero-order, steady-state release of p-nitrophenol. The steady-state reaction obeys Michaelis-Menten kinetics. Comparison of p-nitrophenyl acetate hydrolyses catalyzed by subtilisin and thiol-subtilisin indicates identical kcat/Km, differing kcat and Km, similar pK values of kcat, and different pK values of kcat/Km. The decinnamoylation of subtilisin and thiol-subtilisin derivatives shows similar pK values but the former is much faster than the latter. trans-Cinnamoyl-thiol-subtilisin, however, is much more sensitive toward nucleophiles like glycinamide than is the counterpart of the native enzyme. The hydrolyses of acylamino acid esters and amides are not sensibly catalyzed by thiol-subtilisin. Thiol-subtilisin catalyzes certain hydrolytic reactions by a mechanism similar to that of native subtilisin with the exception that the acyl-enzyme is a thiol ester.

Original languageEnglish
Pages (from-to)610-620
Number of pages11
JournalBiochemistry
Volume6
Issue number2
Publication statusPublished - 1967

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Subtilisin
Sulfhydryl Compounds
Functional groups
Enzymes
Hydrolysis
Serine
Cysteine
Esters
Phenylmethylsulfonyl Fluoride
Nucleophiles
Titration
Amides
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

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The reactivity of thiol-subtilisin, an enzyme containing a synthetic functional group. / Polgár, L.; Bender, Myron L.

In: Biochemistry, Vol. 6, No. 2, 1967, p. 610-620.

Research output: Contribution to journalArticle

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