2af4
From Proteopedia
|
Phosphotransacetylase from Methanosarcina thermophila co-crystallized with coenzyme A
Overview
Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the, acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA, and inorganic phosphate. Two crystal structures of phosphotransacetylase, from the methanogenic archaeon Methanosarcina thermophila in complex with, the substrate CoA revealed one CoA (CoA1) bound in the proposed active, site cleft and an additional CoA (CoA2) bound at the periphery of the, cleft. The results of isothermal titration calorimetry experiments are, described, and they support the hypothesis that there are distinct, high-affinity (equilibrium dissociation constant [KD], 20 microM) and, low-affinity (KD, 2 mM) CoA binding sites. The crystal structures, indicated that binding of CoA1 is mediated by a series of hydrogen bonds, and extensive van der Waals interactions with the enzyme and that there, are fewer of these interactions between CoA2 and the enzyme. Different, conformations of the protein observed in the crystal structures suggest, that domain movements which alter the geometry of the active site cleft, may contribute to catalysis. Kinetic and calorimetric analyses of, site-specific replacement variants indicated that there are catalytic, roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl, of CoA1. The reaction is hypothesized to proceed through base-catalyzed, abstraction of the thiol proton of CoA by the adjacent and invariant, residue Asp316, followed by nucleophilic attack of the thiolate anion of, CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310, binds acetyl phosphate and orients it for optimal nucleophilic attack. The, hypothesized mechanism proceeds through a negatively charged transition, state stabilized by hydrogen bond donation from Ser309.
About this Structure
2AF4 is a Single protein structure of sequence from Methanosarcina thermophila with COA as ligand. Active as Phosphate acetyltransferase, with EC number 2.3.1.8 Full crystallographic information is available from OCA.
Reference
Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila., Lawrence SH, Luther KB, Schindelin H, Ferry JG, J Bacteriol. 2006 Feb;188(3):1143-54. PMID:16428418
Page seeded by OCA on Wed Nov 21 08:05:26 2007
