STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF SUBSTITUTIONS AT THE TYROSINE-55 LYSINE-104 HYDROGEN-BOND IN ESCHERICHIA-COLI INORGANIC PYROPHOSPHATASE

Tyrosine 55 and lysine 104 are evolutionarily conserved residues that form a hydrogen bond in the active site of Escherichia coli inorganic pyrophosphatase (E-PPase). Here we used site-directed mutagenesis to e xamine their roles in structure stabilization and catalysis. Though th ese residues are not part of the subunit interface, Y55F and K104R (bu t not K104I) substitutions markedly destabilize the hexameric structur e, allowing dissociation into active trimers on dilution. A K104I vari ant is nearly inactive while Y55F and K104R variants exhibit appreciab le activity and require greater concentrations of Mg2+ and higher pH f or maximal activity. The effects on activity are explained by (a) incr eased pK(a)s for the catalytically essential base and acid at the acti ve site, (b) decreases in the rate constant for substrate (dimagnesium pyrophosphate) binding to enzyme-Mg-2 complex vs enzyme-Mg-3 complex, and (c) parallel decreases in the catalytic constant for the resultin g enzyme-Mg-2-substrate and enzyme-Mg-3-substrate complexes. The resul ts are consistent with the major structural roles of Tyr55 and Lys104 in the active site. The microscopic rate constant for PPi hydrolysis o n either the Y55F or K104R variants increases, by a factor of 3-4 in t he pH range 7.2-8.0, supporting the hypothesis that this reaction step depends on an essential base within the enzyme active site.