期刊论文详细信息
FEBS Letters
Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding
Avaeva, Svetlana M.2  Kurilova, Svetlana A.2  Nazarova, Tatjana I.2  Rodina, Elena V.2  Vorobyeva, Natalya N.1 
[1] Chemistry Department, Moscow State University, Vorobeyevy Gory, Moscow 119899, Russian Federation;A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobeyevy Gory, Moscow 119899, Russian Federation
关键词: Inorganic pyrophosphatase;    Site-directed mutagenesis;    Metal binding center;    Differential spectrophotometry;   
DOI  :  10.1016/0014-5793(96)00791-0
学科分类:生物化学/生物物理
来源: John Wiley & Sons Ltd.
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【 摘 要 】

Asp-42 located in the active site of E. coli inorganic pyrophosphatase (PPase) has been substituted by Asn by site-directed mutagenesis. This resulted in a 3-fold increase in hydrolytic activity measured under optimal conditions, a 15.5-fold increase in the K m value and retention of the pK values of groups for enzyme and enzyme-substrate complex. The active site of the enzyme contains 4 metal binding centers (I–IV) [Harutyunyan et al. (1996) Eur. J. Biochem., in press]. Asp-42 is located near centers II and IV. The D42N replacement had no effect on Mg2+ binding with center II. At the same time, occupation of center IV eliminates the inhibition of inorganic pyrophosphate hydrolysis by high Mg2+ concentrations typical of wild-type PPase. It is proposed that the increase in activity and decrease in affinity for substrate of the D42N PPase results from changes in Mg2+ binding to center IV. The Mg2+ binding centers of E. coli PPase are lined up in filling order.

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