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

• Published in: FEBS letters 1996-08, Vol.392 (2), p.91-94
• Main Authors: Avaeva, Svetlana M., Rodina, Elena V., Kurilova, Svetlana A., Nazarova, Tatjana I., Vorobyeva, Natalya N.
• Format: Article
• Language: English
• Subjects: Asparagine - genetics; Aspartic Acid - genetics; Catalysis; Differential spectrophotometry; Escherichia coli - enzymology; Escherichia coli - genetics; Fluorides - pharmacology; Hydrolysis; Inorganic Pyrophosphatase; Magnesium - metabolism; Metal binding center; Mutagenesis, Site-Directed; Pyrophosphatases - antagonists & inhibitors; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; Site-directed mutagenesis; Substrate Specificity
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(96)00791-0

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.