EFFECT OF D42N SUBSTITUTION IN ESCHERICHIA-COLI INORGANIC PYROPHOSPHATASE ON CATALYTIC ACTIVITY AND MG2+ BINDING

Asp-42 located in the active site of E. coli inorganic pyrophosphatase (PPase) has been substituted by Asn by site-directed mutagenesis. Thi s resulted in a 3-fold increase in hydrolytic activity measured under optimal conditions, a 15.5-fold increase in the K-m value and retentio n 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 loca ted 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 eli minates the inhibition of inorganic pyrophosphate hydrolysis by high M g2+ concentrations typical of wild-type PPase, It is proposed that the increase in activity and decrease in affinity for substrate of the D4 2N PPase results from changes in Mg2+ binding to center IV. The Mg2+ b inding centers of E. coli PPase are lined up in filling order.