Modulation of Dimer Stability in Yeast Pyrophosphatase by Mutations at the Subunit Interface and Ligand Binding to the Active Site

Anu Salminen,Alexey N Parfenyev,Krista Salli,Irina S Efimova,Natalia N Magretova,Adrian Goldman,Alexander A Baykov,Reijo Lahti

doi:10.1074/jbc.m200101200

Abstract

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > or = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.

Highlights

Phate and phosphate [1, 2]
The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity
Our results indicate that mutation of Trp52 has the most significant effect on dimerization and that active site ligands enhance dimer stability

Summary

Introduction

Phate and phosphate [1, 2]. Due to its relatively simple structure and high catalytic efficiency (kcat/Km ϭ ϳ109 MϪ1 sϪ1), PPase has become a paradigm for mechanistic and structural studies of enzymatic phosphoryl transfer from phosphoric acid anhydrides to water [3, 4]. I PPases are homohexamers of ϳ20-kDa subunits in prokaryotes and homodimers of ϳ32-kDa subunits in eukaryotes with highly conserved active sites and mechanisms of action [3, 4]. Family II members have yet to be characterized in detail, available data suggest that the active sites of family I and family II PPases are quite similar, presenting a remarkable example of convergent enzyme evolution [9, 10]. The active site and subunit interface are separated by about 5 Å [11,12,13] and do not share common amino acid residues. Core intersubunit contact is formed by a three-layer stacking of the aromatic rings of Trp, His, His87Ј, and Trp52Ј, with His and His87Ј forming the central layer (Ј represents residues of the second subunit). Our results indicate that mutation of Trp has the most significant effect on dimerization and that active site ligands enhance dimer stability

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