Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding

FEBS Letters

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.

【 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.

【 授权许可】

Unknown