HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fushimi, N. Articles by Ichishima, E. Search for Related Content PubMed PubMed Citation Articles by Fushimi, N. Articles by Ichishima, E. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 34, 24195-24201, August 20, 1999

Aspzincin, a Family of Metalloendopeptidases with a New Zinc-binding Motif
IDENTIFICATION OF NEW ZINC-BINDING SITES (His¹²⁸, His¹³², and Asp¹⁶⁴) AND THREE CATALYTICALLY CRUCIAL RESIDUES (Glu¹²⁹, Asp¹⁴³, and Tyr¹⁰⁶) OF DEUTEROLYSIN FROM ASPERGILLUS ORYZAE BY SITE-DIRECTED MUTAGENESIS

Naoya Fushimi, Ch'ng Ewe Ee, Tasuku Nakajima, and Eiji Ichishima^§

From the  Laboratory of Molecular and Cellular Biology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan and the ^§ Department of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His¹²⁸ and His¹³² with Arg, of Glu¹²⁹ with Gln or Asp, of Asp¹⁴³ with Asn or Glu, of Asp¹⁶⁴ with Asn, and of Tyr¹⁰⁶ with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His¹²⁸, His¹³², and Asp¹⁶⁴ provide the Zn²⁺ ligands of the enzyme according to a ⁶⁵Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu¹²⁹, Asp¹⁴³, and Tyr¹⁰⁶ are catalytically crucial residues in the enzyme. Glu¹²⁹ may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn²⁺ metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Y. Doi, H. Akiyama, Y. Yamada, C. E. Ee, B. R. Lee, M. Ikeguchi, and E. Ichishima Thermal stabilization of penicillolysin, a thermolabile 19 kDa Zn2+-protease, obtained by site-directed mutagenesis Protein Eng. Des. Sel., March 1, 2004; 17(3): 261 - 266. [Abstract] [Full Text] [PDF]