Site-directed mutagenesis of cysteine to threonine in Proteus vulgaris urease active site increases enzyme activity and stability

Cysteine-319 belongs to the flexible flap at the active site of Proteus vulgaris urease. Replacing this cysteine by threonine resulted in a 20-fold increase of specific activity. Temperature stability increased, susceptibility to inhibition by dipyridyl disulfide decreased, and pH optimum shifted fr...

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Published inBiotechnology letters Vol. 23; no. 15; pp. 1263 - 1267
Main Authors JOSE, Joachim, LAUTER, Sven, STEIN, Martin A
Format Journal Article
LanguageEnglish
Published Dordrecht Springer 01.08.2001
Springer Nature B.V
Subjects
Biological and medical sciences
Biotechnology
Enzymatic activity
Enzymes
Fundamental and applied biological sciences. Psychology
Methods. Procedures. Technologies
phenylmethylsulfonyl fluoride
Protein engineering
Proteins
Proteus vulgaris
toluene fluoride
Enzymatic activity
Enzyme
Active site
Site directed mutagenesis
Bacteria
Hydrolases
Urease
Proteus vulgaris
Thermal stability
Enterobacteriaceae
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Summary:Cysteine-319 belongs to the flexible flap at the active site of Proteus vulgaris urease. Replacing this cysteine by threonine resulted in a 20-fold increase of specific activity. Temperature stability increased, susceptibility to inhibition by dipyridyl disulfide decreased, and pH optimum shifted from 8 to 6.9. K^sub m^ (35 to 12 mM) and V^sub max^ (47.4 to 1.8 μmol min^sup -1^) were substancially altered. Both variants of the enzyme were irreversibly inhibited by phenylmethanesulfonyl fluoride.[PUBLICATION ABSTRACT]
Bibliography:ObjectType-Article-2
SourceType-Scholarly Journals-1
ObjectType-Feature-1
content type line 23
ISSN:0141-5492
1573-6776
DOI:10.1023/A:1010541513179