Reciprocal effects of substitutions at the subunit interfaces in hexameric pyrophosphatase of Escherichia coli. Dimeric and monomeric forms of the enzyme.

A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His(136) and His(140) in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytiä, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734-740). Here we demonstrate that additional substitutions of Tyr(77) and Gln(80) in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg(2+) concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 10(6)-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg(2+)-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting "conformational strain," hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr(77), Gln(80), and His(136) exhibits positive cooperativity in catalysis, consistent with this model.

Trimeric inorganic pyrophosphatase of Escherichia coli obtained by directed mutagenesis.

Escherichia coli inorganic pyrophosphatase is a tight hexamer of identical subunits. Replacement of both His136 and His140 by Gln in the subunit interface results in an enzyme which is trimeric up to 26 mg/mL enzyme concentration in the presence of Mg2+, allowing direct measurements 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 to one of the three sites per monomer than it does to the equivalent site in the hexamer, suggesting this site to be located in the trimer:trimer interface. The otherwise unobtainable hexameric variant enzyme readily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into medium lacking magnesium phosphate or pyrophosphate. The kcat values are similar for the variant trimer and hexamer, but Km values are 3 orders of magnitude lower for the hexamer. Thus, while stabilizing hexamer, the two His residues, per se, are not absolutely required for active-site structure rearrangement upon hexamer formation. The reciprocal effect of hexamerization and product binding to the active site is explained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.