Role of arginines in coenzyme A binding and catalysis by the phosphotransacetylase from Methanosarcina thermophila.
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.
Iyer
P P
PP
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500, USA.
Ferry
J G
JG
D001120:Arginine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D003931:Diacetyl / Q000494:pharmacology; D014644:Genetic Variation; D007700:Kinetics; D017020:Methanosarcina / Q000187:drug effects / Q000201:enzymology*; D015394:Molecular Structure; D010709:Phosphate Acetyltransferase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
C035999:desulfo-coenzyme A; D001120:Arginine; D010709:Phosphate Acetyltransferase; D003931:Diacetyl; D003065:Coenzyme A