PMID:8772181
| Citation |
Avaeva, SM, Rodina, EV, Kurilova, SA, Nazarova, TI and Vorobyeva, NN (1996) Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding. FEBS Lett. 392:91-4 |
|---|---|
| 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. |
| Links | |
| Keywords |
Asparagine/genetics; Aspartic Acid/genetics; Catalysis; Escherichia coli/enzymology; Escherichia coli/genetics; Fluorides/pharmacology; Hydrolysis; Inorganic Pyrophosphatase; Magnesium/metabolism; Mutagenesis, Site-Directed; Pyrophosphatases/antagonists & inhibitors; Pyrophosphatases/genetics; Pyrophosphatases/metabolism; Substrate Specificity |
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