STRUCTURE AND FUNCTION-ANALYSIS OF ESCHERICHIA-COLI INORGANIC PYROPHOSPHATASE - IS A HYDROXIDE ION THE KEY TO CATALYSIS

Using site-directed mutagenesis, we have completed replacing all 17 pu tative active site residues of Escherichia coil inorganic pyrophosphat ase (PPase). We report here the production of 11 new variant proteins and their initial characterization, including thermostability, hydroph obicity, oligomeric structure, and specific activity at pH 8. Studies of the pH-rate profiles of 12 variants containing substitutions for po tentially essential residues showed that the effect of the mutation wa s always to increase the pK(a) of a basic group essential for both sub strate binding and catalysis by 1-3 pH units. The D70E variant had the lowest activity at all pHs; the K29R, R43K, and K142R variants also h ad low k(cat)/K-m values. The principal effect seen in the other varia nt proteins was higher and sharper pH optima; their pH-independent k(c at) and k(cat)/K-m values changed at most by a factor of 8. Our result s suggest that the most likely candidate for the essential basic group affected by all mutations in the active site is a hydroxide ion stabi lized by coordination to the essential Mg2+ ions. Analyzing our result s using the structure recently obtained for E. coli PPase [Kankare et al. (1994) Protein Eng. 7, 823-830] led us to identify a group of resi dues, centered around Asp70 and including Tyr55, Asp65, Asp67, Asp102, and Lys104, that we believe binds the magnesium ions that are critica l for the activity, possibly by stabilizing the essential hydroxide. O thers, including Lys29, Arg43, and Lys142, are more spread out and mor e positively charged. They appear to be involved in binding substrate and product. Tyr55 is also a key part of the hydrophobic core of E. co li PPase; when it or residues that interact with it are conservatively mutated, there are changes in the overall structure of the enzyme as assayed by thermostability, hydrophobicity, or oligomeric structure.