Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.
Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding.
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Laboratory of Bacterial Toxins, Division of Bacterial Products, OVRR, CBER, FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA.
Zapata
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Picone
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Vann
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D003570:Cytidine Triphosphate / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000216:N-Acylneuraminate Cytidylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics
D011993:Recombinant Fusion Proteins; D011732:Pyridoxal Phosphate; D003570:Cytidine Triphosphate; D001120:Arginine; D000216:N-Acylneuraminate Cytidylyltransferase; D008239:Lysine