Allosteric and Catalytic Functions of the PPi-binding Motif in the ATP Sulfurylase-GTPase System

Daniel E Pilloff,Thomas S Leyh

doi:10.1074/jbc.m306897200

Daniel E Pilloff, Thomas S Leyh

Open Access

https://doi.org/10.1074/jbc.m306897200

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Abstract

ATP sulfurylase, from Escherichia coli K-12, catalyzes and couples the Gibbs potentials of two reactions, GTP hydrolysis and activated sulfate (APS, adenosine 5'-phosphosulfate) synthesis. Coupling these potentials requires that the catalytic cycle include reaction stage-dependent conformational changes that gate the activities of the two active sites. These interactions were probed in a mutagenesis study of a highly conserved pyrophosphate-binding motif (SXGXDS), which is located at the APS-forming active site. The motif appears to be unique to the N-type PPi synthetase family, and mutations in it are linked, in other systems, to citrullinemia, an often fatal orphan disease. The conserved sites in the motif were evaluated individually for their ability to activate GTP hydrolysis (which reports interactions among the activator (AMP or Mg2+.PPi), the enzyme, and GTP), to affect the energetic coupling of the two reactions, and to alter the kinetic constants of the adenylyl transfer reaction in the absence of guanine nucleotide. What emerges from this first mutagenic exploration of the PPi motif in any adenylyltransferase is that the residues of the motif participate differently, and in sometimes profoundly important ways, in the different functions of the enzyme.

Highlights

The native enzyme is a tetramer of heterodimers, each of which is composed of a GTPase subunit (CysN, 53 kDa) and an adenylyltransferase subunit (CysD, 23 kDa) [3, 4]
ATP sulfurylase, from Escherichia coli K-12, catalyzes and couples the Gibbs potentials of two reactions, GTP hydrolysis and activated sulfate (APS, adenosine 5؅phosphosulfate) synthesis. Coupling these potentials requires that the catalytic cycle include reaction stage-dependent conformational changes that gate the activities of the two active sites. These interactions were probed in a mutagenesis study of a highly conserved pyrophosphate-binding motif (SXGXDS), which is located at the APS-forming active site
As ATP sulfurylase moves through the various stages of its catalytic cycle, the ground and transition state energetics of the GTPase activity are modulated by inorganic pyrophosphate in ways that depend upon the role that PPi is asked to play

Summary

Introduction

The native enzyme is a tetramer of heterodimers, each of which is composed of a GTPase subunit (CysN, 53 kDa) and an adenylyltransferase subunit (CysD, 23 kDa) [3, 4]. As ATP sulfurylase moves through the various stages of its catalytic cycle, the ground and transition state energetics of the GTPase activity are modulated by inorganic pyrophosphate in ways that depend upon the role that PPi is asked to play. When it is the sole activator of GTP hydrolysis, PPi stimulates GTPase turnover 20-fold over the basal or non-activated level (0.010 sϪ1 [17]), and the interaction energies that accelerate catalysis occur predominantly downstream of the partially rate-limiting isomerization that precedes scission of the ␤,␥bond (18 –21). The functions of each of the conserved residues of the motif in adenylyl transfer and allosteric interactions between the APS-forming and GTPase active sites were explored in the current investigation through subtle mutagenic alterations that revealed that the roles of the residues change with the catalytic demands placed upon them

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