NDLI: Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

Content Provider	PubMed Central
Author	Kobayashi, Michihiko Fujiwara, Yoshie Goda, Masahiko Komeda, Hidenobu Shimizu, Sakayu
Copyright Year	1997
Abstract	Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 → Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 → Asn substitution as well as Ser195 → Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.
Starting Page	11986
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	22
Volume Number	94
Language	English
Publisher	The National Academy of Sciences of the USA
Publisher Date	1997-10-28
Access Restriction	Open
Rights Holder	The National Academy of Sciences of the USA
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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