Reciprocal effects of substitutions at the subunit interfaces in hexamericpyrophosphatase 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 monomer s. Earlier we reported that substitution of His(136) and His(140) in the in tertrimeric subunit interface splits the molecule into active trimers (Veli chko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytia, T., Go ldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemist ry 37, 734-740). Here we demonstrate that additional substitutions of Tyr(7 7) and Gln(80) in the intratrimeric interface give rise to moderately activ e dimers or virtually inactive monomers, depending on pH, temperature, and Mg2+ concentration. Successive dissociation of the hexamer into trimers, di mers, and monomers progressively decreases the catalytic efficiency (by 10( 6)-fold in total), and conversion of a trimer into dimer decreases the affi nity of one of the essential Mg2+-binding sites/monomer. Disruptive substit utions predominantly in the intratrimeric interface stabilize the intertrim eric interface and vice versa, suggesting that the optimal intratrimeric in teraction is not compatible with the optimal intertrimeric interaction. Bec ause of the resulting "conformational strain," hexameric wild-type structur e appears to be preformed to bind substrate. A hexameric triple variant sub stituted at Tyr(77), Gln(80), and His(136) exhibits positive cooperativity in catalysis, consistent with this model.