Identification of Cys-150 in the active site of phosphomannose isomerase from Candida albicans.
Candida albicans phosphomannose isomerase (PMI) (EC 5.3.1.8) has been recently cloned and overexpressed in Escherichia coli. The enzyme can be irreversibly inactivated by iodoacetate in 50 mM borate buffer, pH 9.0, in a time-dependent manner at a rate of 4.2 +/- 0.03 min-1 M-1. This inhibition can be prevented by the substrate mannose 6-phosphate with a Ks of 0.22 +/- 0.05 mM, slightly lower than its Km value. However, metals such as zinc and cadmium, which are reversible, competitive inhibitors for PMI, do not protect the enzyme against modification. The protein has been labeled by using [2-14C]iodoacetate, in the presence or absence of substrate, and the protein is fully inactivated when 1.0 thiol group is modified per molecule of enzyme. Tryptic maps of the modified protein have been produced. The protected peptide has been identified and sequenced, and the phenylthiohydantoin amino acids have been collected. The modified amino acid is Cys-150. This cysteine residue is conserved in mammalian and yeast phosphomannose isomerases, but not in bacterial species where it is replaced with asparagine. We therefore purified PMI from E. coli and showed that this enzyme is not sensitive to inactivation by iodoacetate. The iodoacetate is presumably inhibiting PMI by sterically blocking the mannose 6-phosphate binding site. Multiple sequence alignment procedures were used to try to identify potential ligands of the zinc atom that is essential for enzyme activity and thus to delineate the active site region.(ABSTRACT TRUNCATED AT 250 WORDS)
Coulin
F
F
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Magnenat
E
E
Proudfoot
A E
AE
Payton
M A
MA
Scully
P
P
Wells
T N
TN
D000595:Amino Acid Sequence; D001665:Binding Sites; D002176:Candida albicans / Q000201:enzymology*; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D005656:Fungal Proteins; D007461:Iodoacetates; D008667:Metalloproteins; D010446:Peptide Fragments; D008359:Mannose-6-Phosphate Isomerase; D015032:Zinc; D003545:Cysteine; D019807:Iodoacetic Acid