Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase.
Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.
Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA.
Sandra E
SE
Ríos
Thomas
T
Nowak
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D003848:Deoxyguanine Nucleotides; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C029603:deoxyguanosine triphosphate; D004228:Dithionitrobenzoic Acid; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003545:Cysteine