title: Mechanism of irreversible inactivation of phosphomannose isomerases by silver ions and flamazine.
abstract: Silver ions and silver-containing compounds have been used as topical antimicrobial agents in a variety of clinical situations. We have previously shown that the enzyme phosphomannose isomerase (PMI) is essential for the biosynthesis of Candida albicans cell walls. In this study, we find that PMI can be inhibited by silver ions. This process is shown to be irreversible, and is a two-step process, involving an intermediate complex with a dissociation constant, Ki, of 59 +/- 8 microM, and a maximum rate of inactivation of 0.25 +/- 0.04 min-1 in 50 mM Hepes buffer, pH 8.0 at 37 degrees C. The enzyme can be protected against this inactivation by the substrate mannose 6-phosphate, with a dissociation constant of 0.31 +/- 0.04 mM, close to its Km value. Flamazine (silver sulfadiazine) is a silver-containing antibiotic which is used clinically as a topical antimicrobial and antifungal agent. We compared the ability of silver sulfadiazine and two other silver-containing compounds to irreversibly inactivate C. albicans PMI. The addition of the organic moiety increased the affinity of the compounds, with silver sulfadiazine showing a Ki of 190 +/- 30 nM. In all cases, the maximum inhibition rate was similar, implying a similar rate-determining step. Silver sulfadiazine does not inhibit Escherichia coli PMI, and this suggests a role of the only free cysteine, Cys-150, in the inactivation process. To confirm this, we mutated this residue to alanine in C. albicans PMI. The resultant Cys150 --> Ala mutant protein showed similar Vm and Km values to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Glaxo Institute for Molecular Biology, Geneva, Switzerland.
authors.forename: T N
authors.initials: TN
authors.lastname: Wells
authors.forename: P
authors.initials: P
authors.lastname: Scully
authors.forename: G
authors.initials: G
authors.lastname: Paravicini
authors.forename: A E
authors.initials: AE
authors.lastname: Proudfoot
authors.forename: M A
authors.initials: MA
authors.lastname: Payton
mesh_terms: D000409:Alanine / Q000235:genetics; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D002227:Carbazoles / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D007211:Indoles / Q000494:pharmacology; D008359:Mannose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors* / Q000235:genetics; D008627:Mercuric Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds / Q000494:pharmacology*; D012834:Silver / Q000494:pharmacology*; D012837:Silver Sulfadiazine / Q000494:pharmacology*
chemical_list: D000935:Antifungal Agents; D002227:Carbazoles; D002414:Cations, Monovalent; C094413:GR 131921 X; C094414:GR 132465 X; D007211:Indoles; D009942:Organometallic Compounds; D012834:Silver; D008627:Mercuric Chloride; D008359:Mannose-6-Phosphate Isomerase; D003545:Cysteine; D000409:Alanine; D012837:Silver Sulfadiazine