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Highly conservative sequence in the carboxyl terminus of sarcosine oxidase is important for substrate binding

Authors
Journal
Journal of Fermentation and Bioengineering
0922-338X
Publisher
Elsevier
Publication Date
Volume
84
Issue
6
Identifiers
DOI: 10.1016/s0922-338x(97)81917-4
Keywords
  • Sarcosine Oxidase
  • Conservative Sequence
  • Substrate Binding
Disciplines
  • Biology

Abstract

Abstract The function of the highly conservative sequence —G(344)-F-S-G-H-G-F-K-F(352)— in the carboxyl terminus of sarcosine oxidase was investigated using site-directed mutagenesis. When H-348 was substituted with uncharged amino acids, the K m values of sarcosine oxidase markedly increased, although the k cat values remained the same as that of the wild-type enzyme. The kinetic parameters obtained with other mutations also suggested that the conservative sequence acts as the substrate-binding site. When K-351 was replaced by Ala, the mutant K351A could not bind the coenzyme FAD (flavin adenine dinucleotide). This result suggested that K-351 interacts with the FAD-binding site of the amino-terminal region. The enzymic activities of the mutants H348Q and H348A were lost at neutral and acidic pH as a result of the disappearance of the positive charge on H-348.

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