Arginine 304 is an active site residue in phosphomannose isomerase from Candida albicans.
The reaction catalyzed by Candida albicans phosphomannose isomerase (PMI) (EC 5.3.1.8) has a bell-shaped pH dependence, with pKa's at 5.6 and 8.7. The enzyme can be inhibited in a time-dependent manner using the arginine-specific modification reagent phenylglyoxal. This modification takes place with a rate constant of 0.022 +/- 0.002 min-1 mM-1 at 37 degrees C in 50 mM Hepes buffer, pH 8.5. The enzyme can be protected from this inactivation by the addition of the substrate mannose 6-phosphate at concentrations close to its Km value. The pH dependence of the inactivation reaction shows a single pKa at 9.1 +/- 0.1, which is close to one of the values for the pH dependence of the enzyme-catalyzed reaction. Using [7-14C]phenylglyoxal, it is shown that a single molecule is incorporated into the enzyme in the absence of substrate and that this inactivates the enzyme. This incorporation of radioactivity is prevented by the coincubation with substrate. The modified protein has then been reduced with sodium borohydride to fix the modification and then cleaved with Asp-N protease. The resultant peptides were separated by HPLC, and the radioactivity was counted. Sequencing of the peptide with the highest incorporation level identified it as DNVVRAGFTPKFK, which corresponds to amino acids 300-312 of phosphomannose isomerase. Radioactive counting of the phenylthiohydantoin amino acid derivatives confirmed that the modified amino acid was arginine 304. The role of this residue in the catalytic reaction of phosphomannose isomerase is discussed.
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Glaxo Institute for Molecular Biology, Geneva, Switzerland.
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D000595:Amino Acid Sequence; D001120:Arginine / Q000032:analysis / Q000378:metabolism*; D001665:Binding Sites; D002176:Candida albicans / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010658:Phenylglyoxal / Q000494:pharmacology
D001120:Arginine; D008359:Mannose-6-Phosphate Isomerase; D010658:Phenylglyoxal