title: Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding.
abstract: 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 Km 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.
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation.
authors.forename: S M
authors.initials: SM
authors.lastname: Avaeva
authors.forename: E V
authors.initials: EV
authors.lastname: Rodina
authors.forename: S A
authors.initials: SA
authors.lastname: Kurilova
authors.forename: T I
authors.initials: TI
authors.lastname: Nazarova
authors.forename: N N
authors.initials: NN
authors.lastname: Vorobyeva
mesh_terms: D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005459:Fluorides / Q000494:pharmacology; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D008274:Magnesium / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001224:Aspartic Acid; D001216:Asparagine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D005459:Fluorides