| In almost all fermentative eubacteria, acetate kinase and phosphotransacetylase catalyze the last two steps in anaerobic fermentation to acetate. In acetate-consuming archaea from the Methanosarcina genus, these enzymes catalyze the first two steps in the anaerobic fermentation of acetate to methane. Acetate kinase and phosphotransacetylase from Methanosarcina thermophila share high sequence similarity with the respective enzymes from other procaryotes, and have served as models to study the structure and catalytic mechanism of each. Neither acetate kinase nor phosphotransacetylase is present in Eucarya or most species of Archaea where acetate metabolism is managed by the single enzyme acetyl-CoA synthetase, which bears no homology to either of the former.;An overview of acetate metabolism by Methanosarcina sp. is presented in Chapter 1, and the biochemical histories of acetate kinase and phosphotransacetylase are discussed in detail in Chapter 2. This thesis contains four experimental sections which address structural and mechanistic questions about both enzymes. Chapter 3 describes the structural identification and biochemical characterization of the acetate binding pocket of M. thermophila acetate kinase. Molecular modeling and biochemical analyses of site-specific replacement variants with perturbations to the pocket formed by residues Val93, Leu122, Phe 179, and Pro232 revealed that the size of the pocket dictates the specificity of the enzyme for carboxylic acids. Chapter 4 presents an improved expression and purification protocol for M. thermophila phosphotransacetylase, along with a steady-state kinetic analysis that supports a ternary complex kinetic mechanism for the enzyme. Chapter 5 introduces the crystal structure of M. thermophila Pta, which revealed a homodimeric enzyme with each monomer composed of two alpha/beta domains with a cleft at the domain boundary proposed to contain the active site. Two dimers were present in each asymmetric unit, and a comparison of the four monomers revealed substantial variations in the geometry of the cleft. The structure does not belong to any of the previously defined structural superfamilies, and a homology search revealed NADP +-dependent citrate and isopropylmalate dehydrogenases as the only homologs with similar two-domain architecture. Two crystal structures of M. thermophila Pta in complex with CoA are presented in Chapter 6. The structures and a calorimetric analysis of CoA binding indicated two CoA binding sites per Pta monomer: a proposed catalytic site, and a secondary binding site of unknown function. Kinetic and calorimetric analyses of site-specific replacement variants indicated catalytic roles for Ser309 and Arg310 that reside proximal to the reactive sulfhydryl of the CoA bound in the proposed catalytic site. The reaction is hypothesized to proceed through base-catalyzed abstraction of the sulfhydryl proton of CoA by the adjacent and invariant Asp316 followed by nucleophilic attack of the CoA thiolate anion on the carbonyl carbon of acetyl phosphate. Arg310 is proposed to bind acetyl phosphate and orient it for optimal nucleophilic attack. The hypothesized mechanism proceeds through formation of a negatively charged transition state stabilized by hydrogen bond donation from Ser309. |
