Role of Arginines in Coenzyme A Binding and Catalysis by the Phosphotransacetylase from Methanosarcina thermophila

Abstract

Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH 3 COOPO 3 2؊ ؉ CoASH % CH 3 COSCoA ؉ HPO 4 2؊ . The role of arginine residues was investigated for the phosphotransacetylase from Methanosarcina thermophila. Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA. Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3-phosphate of CoA. Arg 133 is postulated to interact with the 5-phosphate of CoA. Large decreases in k cat and k cat /K m for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis. Large decreases in k cat and k cat /K m were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity. Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding. Chemical modification of the wild-type enzyme by 2,3-butanedione and substrate protection by CoA indicated that at least one reactive arginine is in the active site and is important for activity. The inhibition pattern of the R87Q variant indicated that Arg 87 is modified, which contributes to the inactivation; however, at least one additional active-site arginine is modified leading to enzyme inactivation, albeit at a lower rate.Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH 3 COOPO 3 2Ϫ ϩ CoASH % CH 3 COSCoA ϩ HPO 4 2Ϫ . Phosphotransacetylase and acetate kinase are important components of the energy-yielding pathway in most anaerobic microbes from the domain Bacteria. In the archaeon Methanosarcina thermophila, sequential reactions catalyzed by acetate kinase and phosphotransacetylase initiate the pathway of acetoclastic methanogenesis by activating acetate to acetylCoA (1). Though acetyl transfer has been studied in other enzymes, very little is known concerning the catalytic mechanism of phosphotransacetylase. Until recently, the only biochemical study reported was in 1976 on the enzyme from Clostridium kluyveri (2). Genes encoding phosphotransacetylase from at least 34 organisms have been reported and all share 51 to 79% deduced sequence similarity to the M. thermophila enzyme (BLAST search results), suggesting similar mechanisms. Recently, the M. thermophila phosphotransacetylase was overexpressed in Escherichia coli, allowing the use of site-directed mutagenesis to investigate the mechanism (6, 8). Thus, this enzyme can serve as a model for all phosphotransacetylases. Chemical modification studies using N-ethylmaleimide implicated a cysteine as an essential active-site residue in the...

Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from Methanosarcina thermophila

“…3B). The interactions observed for Arg 87 and Arg 133 are in excellent agreement with kinetic analyses of site-specific variants, which predicted that Arg 87 interacts with the 3Ј phosphate and Arg 133 interacts with one of the 5Ј phosphates (12). Atoms N-1 and N-6 of the adenine base in CoA 2 were observed to hydrogen bond to Ala 148 in both monomers, and these interactions were the only hydrogen bond interactions observed between CoA 2 and monomer B. N-6 is also close (ϳ4.2 and 4.8 Å in monomers A and B, respectively) to the S atom of Met 174 , the same residue which also interacts with N-6 of CoA (Fig.…”

“…Both binding events were characterized by negligible entropic contributions. Based on interactions observed in the cocrystallized and soaked structures, it can be assumed that the higher-affinity binding site corresponds to (12), the reactive sulfhydryl of CoA 2 is pointed out toward the solvent (Fig. 3B).…”

“…Cys 312 was predicted to be present in the active site, although it is not essential for catalysis (31). Arg 87 and Arg 133 were proposed to interact with the 3Ј and 5Ј phosphate groups of CoA, respectively (12), while Arg 310 was found to be essential for catalysis, although its role was not defined further (31). In spite of the insight gained from the site-specific replacement studies, key questions remained.…”

In Vivo and in Vitro Analyses of Single-amino Acid Variants of the Salmonella enterica Phosphotransacetylase Enzyme Provide Insights into the Function of Its N-terminal Domain

“…Perhaps in some habitats the use of nonregulated Pta I enzyme is favored over the allosterically controlled Pta II enzyme, because the concentration of acetate in the environment is low. This idea is supported by reported kinetic analyses of Pta I enzymes, whose substrate affinity constants are at least 1 order of magnitude lower than Pta II enzymes and are also faster than Pta II enzymes (26,65). Whether a prokaryote uses a Pta I (not allosterically regulated) or a Pta II (allosterically regulated) class enzyme may hinge on the existence of an alternative mechanism for modulating the level of Pta activity (e.g.…”

Structural and Kinetic Analyses of Arginine Residues in the Active Site of the Acetate Kinase from Methanosarcina thermophila

“…The enzyme also plays an essential role in the fermentation of acetate to methane, which accounts for most of the one billion metric tons of methane produced annually from the decomposition of organic matter by anaerobic microbial consortia (3). In Methanosarcina thermophila, acetate kinase catalyzes the first step in the pathway by activating acetate to acetyl phosphate prior to transfer of the acetyl moiety to CoA catalyzed by phosphotransacetylase (4,5). In later steps of the pathway, the acetyl moiety is further metabolized to methane and carbon dioxide (6).…”