DISSOCIATION OF HEXAMERIC ESCHERICHIA-COLI INORGANIC PYROPHOSPHATASE INTO TRIMERS ON HIS-136-]GLN OR HIS-140-]GLN SUBSTITUTION AND ITS EFFECT ON ENZYME CATALYTIC PROPERTIES

Each of the five histidines in Escherichia coli inorganic pyrophosphat ase (PPase) was replaced in turn by glutamine. Significant changes in protein structure and activity were observed in the H136Q and H140Q va riants only. In contrast to wild-type PPase, which is hexameric, these variants can be dissociated into trimers by dilution, as shown by ana lytical ultracentrifugation and cross-linking. Mg2+ and substrate stab ilize the hexameric forms of both variants. The hexameric H136Q- and H 140Q-PPases have the same binding affinities for magnesium ion as wild -type, but their hydrolytic activities under optimal conditions are, r espectively, 225 and 110% of wild-type PPase, and their synthetic acti vities, 340 and 140%. The increased activity of hexameric H136Q-PPase results from an increase in the rate constants governing most of the c atalytic steps in both directions. Dissociation of the hexameric H136Q and H140Q variants into trimers does not affect the catalytic constan ts for PPi hydrolysis between pH 6 and 9 but drastically decreases the ir affinities for Mg2PPi and Mg2+. These results prove that His-136 an d His-140 are key residues in the dimer interface and show that hexame r formation improves the substrate binding characteristics of the acti ve site.