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Identification of Cys-150 in the active site of phosphomannose isomerase from Candida albicans
F Coulin1, E Magnenat, A E Proudfoot
1Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Abstract:
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)
Insights
Candida albicans phosphomannose isomerase (PMI) is inactivated by iodoacetate, with Cys-150 being the modified residue. Bacterial PMI is not affected, suggesting steric blocking of the active site.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Candida albicans phosphomannose isomerase (PMI) is crucial for mannose metabolism.
- Understanding PMI's active site is key to developing targeted inhibitors.
Purpose of the Study:
- To elucidate the mechanism of iodoacetate inactivation of C. albicans PMI.
- To identify the specific residue modified by iodoacetate.
- To investigate the role of Cys-150 in enzyme activity and active site structure.
Main Methods:
- Enzyme kinetics and inhibition studies with iodoacetate and mannose 6-phosphate.
- Protein labeling with [2-14C]iodoacetate.
- Tryptic peptide mapping, sequencing, and amino acid analysis.
- Multiple sequence alignment of PMI from various species.
Main Results:
- PMI irreversibly inactivated by iodoacetate (rate 4.2 min-1 M-1).
- Inhibition prevented by mannose 6-phosphate (Ks = 0.22 mM).
- Cys-150 is the modified residue, with 1.0 thiol group modification per enzyme molecule.
- Bacterial PMI from E. coli is resistant to iodoacetate, indicating Cys-150 is specific to yeast/mammalian enzymes.
Conclusions:
- Iodoacetate inactivates C. albicans PMI by modifying Cys-150.
- This modification likely causes steric hindrance at the mannose 6-phosphate binding site.
- Cys-150 is a key residue in the active site of fungal and mammalian PMI, but not bacterial PMI.