PMID: 10162Sep 15, 1976

Kinetic investigation of the staphylococcal protease-catalyzed hydrolysis of synthetic substrates

European Journal of Biochemistry
J Houmard


In investigating the staphylococcal protease-catalyzed hydrolysis of N-tert-butoxycarbonyl-L-glutamate alpha-phenyl ester, N-benzyloxycarbonyl-L-glutamate alpha-phenyl ester and N-benzyloxycarbonyl-L-glutamate alpha-p-nitroanilide, we obtained kinetic evidence consistent with the formation of an acyl-enzyme intermediate. We found that addition of a nucleophile, such as methanol, led to the partition of the common acyl-enzyme intermediate between water and the alcohol. With N-benzyl-oxycarbonyl-L-glutamate alpha-phenyl ester, a specific ester substrate, deacylation was shown to be the rate-limiting step. By studying the kcat/Km ratio of these hydrolyses as a function of pH, we have shown that two ionizable groups on the enzyme are essential to the catalytic process. One of these groups has a pK of 6.58 and the other, a pK of 8.25. The assignment of these pK values is discussed in connection with the known features of the serine proteinase reaction mechanism. In addition, monovalent anions were shown to inhibit staphylococcal protease hydrolyses. They seem to compete with the negative charge of the substrate, thus inhibiting its binding on the enzyme molecule. Finally we compared the kinetic parameters obtained with five protease...Continue Reading


Jan 1, 1976·International Journal of Peptide and Protein Research·J Houmard
May 2, 1974·European Journal of Biochemistry·A C RydénL Philipson
Dec 1, 1972·Proceedings of the National Academy of Sciences of the United States of America·J Houmard, G R Drapeau
Dec 29, 1974·Biochimica Et Biophysica Acta·A Bjoörklind, H Jörnvall
Jun 15, 1974·FEBS Letters·M Philipp, M L BENDER
Jan 1, 1970·Annual Review of Biochemistry·D M Blow, T A Steitz
Jan 18, 1969·Nature·C S WrightJ Kraut
Jun 20, 1965·Journal of the American Chemical Society·J R WHITAKER, M L BENDER
Jan 1, 1965·Annual Review of Biochemistry·M L BENDER, J KEZDY

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