Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila.
Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.
Rasche
M E
ME
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802-4500, USA.
Smith
K S
KS
Ferry
J G
JG
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001421:Bacteria, Anaerobic / Q000201:enzymology; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D017020:Methanosarcina / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010709:Phosphate Acetyltransferase / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011765:Pyruvaldehyde / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D011765:Pyruvaldehyde; D001120:Arginine; D010709:Phosphate Acetyltransferase; D003545:Cysteine; D005033:Ethylmaleimide; D003065:Coenzyme A