TRIMERIC INORGANIC PYROPHOSPHATASE OF ESCHERICHIA-COLI OBTAINED BY DIRECTED MUTAGENESIS

Escherichia coli inorganic pyrophosphatase is a tight hexamer of ident ical subunits. Replacement of both His136 and Kis140 by Gin in the sub unit interface results in an enzyme which is trimeric up to 26 mg/mL e nzyme concentration in the presence of Mg2+, allowing direct measureme nts of Mg2+ binding to trimer by equilibrium dialysis. The results of such measurements, together with the results of activity measurements as a function of [Mg2+] and pH, indicate that Mg2+ binds more weakly t o one of the three sites per monomer than it does to the equivalent si te in the hexamer, suggesting this sire to be located in the trimer:tr imer interface. The otherwise unobtainable hexameric variant enzyme re adily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into me dium lacking magnesium phosphate or pyrophosphate. The k(cat) values a re similar for the variant trimer and hexamer, but K-m, values are 3 o rders of magnitude lower for the hexamer. Thus, while stabilizing hexa mer, the two His residues, pet-se, are not absolutely required for act ive-site structure rearrangement upon hexamer formation. The reciproca l effect of hexamerization and product binding to the active site is e xplained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.