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Proton-relay system of carboxypeptidase Y as a sole catalytic site: studies on mutagenic replacement of his 397.

Abstract：

:His397 was replaced with alanine by site-directed mutagenesis of the cloned PRC1 gene in order to confirm the role of this residue in the proton-relay system of carboxypeptidase Y (CPY). The expressed and purified H397A showed a CD spectrum almost identical to that of the wild type enzyme, but its heat stability and conformation on heating differed somewhat. Kinetic analysis showed that the kcat values of the purified H397A toward the peptide substrates, Z-Phe-Leu and Z-Gly-Phe, were reduced to approximately 4 x 10(-5)-fold, whereas the Km values remained almost unchanged. The activity of the H397A preparation with the ester substrate, Ac-Phe-OEt, was negligible. The low activity of our H397A was lost on treatment with DFP and Z-Phe-CH2Cl, site-specific inhibitors, respectively, for Ser146 and His397, and with the HgCl2 and PCMB, SH-reagents for Cys341. After treatment with these inhibitors, the kcat value for the H397A preparation toward Z-Phe-Leu decreased 1 x 10(3)-fold or more. The value was approximately 10(-8) for the wild type enzyme. This level of activity is 10(3)-fold lower than the reported value for the same mutant of CPY [Carlsberg Res. Commun. 54, 165-171 (1989)], and more than 10-fold lower than the values for the corresponding His-to-Ala mutants of trypsin [J. Am. Chem. Soc. 114, 1784-1790 (1992)] and subtilisin [Nature 332, 564-568 (1988)]. These findings, together with the pH profiles and chromatographic behavior, are evidence that the low activity of the H397A preparation is due to contamination by wild type CPY. The decreased kcat value of our H397A mutant is the lowest reported among the corresponding histidine mutants of serine proteases. We conclude that the proton-relay system composed of Ser146 and His397 is the sole catalytic center of CPY, and that its destruction leads to complete inactivation.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Jung G,Ueno H,Hayashi R

doi

10.1093/oxfordjournals.jbchem.a022133

subject

Has Abstract

pub_date

1998-08-01 00:00:00

pages

446-50

issue

eissn

0021-924X

issn

1756-2651

journal_volume

124

pub_type

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Proton-relay system of carboxypeptidase Y as a sole catalytic site: studies on mutagenic replacement of his 397.