Abstract
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.
Highlights
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
These involve a mixture of hydrophilic and hydrophobic interactions that include Tyr[77] and the backbone NH of Gln[80] (5); the total surface area buried per monomer is about 1300 Å2 (6)
We describe the effects of substitutions of Tyr[77] and Gln[80], predominantly at the intratrimeric interface, on the quaternary structure and catalytic activity of E. coli PPase (E-PPase)
Summary
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. The intratrimer contacts are of a circular “head-to-tail” type, meaning that each monomer has two different intratrimer contact regions (Fig. 1B) (5, 6) These involve a mixture of hydrophilic and hydrophobic interactions that include Tyr[77] and the backbone NH of Gln[80] (5); the total surface area buried per monomer is about 1300 Å2 (6). We describe the effects of substitutions of Tyr[77] and Gln[80], predominantly at the intratrimeric interface, on the quaternary structure and catalytic activity of E-PPase. This interface mainly consists of hydrophobic contacts between strands and contains few hydrogen bonds between monomers (Fig. 1C). The results of this study shed light on the interactions between different subunit contacts in E-PPase
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