Carboxymethylation of MutS-cysteine-15 specifically inactivates adenosylcobalamin-dependent glutamate mutase. Examination of the role of this residue in coenzyme-binding and catalysis.
The sensitivity of adenosylcobalamin (AdoCbl)-dependent glutamate mutase toward thiol-directed reagents has been investigated. Iodoacetate specifically alkylates one cysteine residue, Cys-15, in MutS with concomitant irreversible loss of enzyme activity. Cys-15 lies between the conserved residues Asp-14 and His-16, that are believed to coordinate cobalt to form a Co-His-Asp hydrogen-bonded "triad" when AdoCbl is bound by the enzyme. Although inactive, carboxymethylated MutS still bound AdoCbl with only a 5-fold increase in apparent Kd. To determine whether Cys-15 plays an essential role in catalysis, it was mutated to serine and to alanine. These mutants were active, but both exhibited decreased Vmax and increased apparent Km and Kd for AdoCbl. To mimic the effect of carboxymethylation, Cys15 was mutated to aspartate and, as an isosteric control, to asparagine. Neither of these mutants was active: MutS-C15N bound AdoCbl approximately 10-fold weaker than wild type, whereas MutS-C15N bound AdoCbl over 100 times less strongly than wild type. The results demonstrate both coenzyme-binding and catalysis to be very sensitive to mutations at position 15 that could potentially perturb the Co-His-Asp hydrogen-bonding network.
Holloway
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DE
Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom.
Chen
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HP
Marsh
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EN
D000251:Adenosine Triphosphatases; D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology; D003038:Cobamides / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D019751:Intramolecular Transferases; D007700:Kinetics; D051719:MutS DNA Mismatch-Binding Protein; D016296:Mutagenesis; D011485:Protein Binding
D001426:Bacterial Proteins; D003038:Cobamides; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D000591:Amino Acid Isomerases; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide; D003545:Cysteine