MECHANISM OF IRREVERSIBLE INACTIVATION OF PHOSPHOMANNOSE ISOMERASES BY SILVER IONS AND FLAMAZINE

Silver ions and silver-containing compounds have been used as topical antimicrobial agents in a variety of clinical situations. We have prev iously shown that the enzyme phosphomannose isomerase (PMI) is essenti al for the biosynthesis of Candida albicans cell walls. In this study, we find that PMI can be inhibited by silver ions. This process is sho wn to be irreversible, and is a two-step process, involving an interme diate complex with a dissociation constant, K-i, of 59 +/- 8 mu M, and a maximum rate of inactivation of 0.25 +/- 0.04 min(-1) in 50 mM Hepe s buffer, pH 8.0 at 37 degrees C. The enzyme can be protected against this inactivation by the substrate mannose 6-phosphate, with a dissoci ation constant of 0.31 +/- 0.04 mM, close to its K-m value. Flamazine (silver sulfadiazine) is a silver-containing antibiotic which is used clinically as a topical antimicrobial and antifungal agent. We compare d the ability of silver sulfadiazine and two other silver-containing c ompounds to irreversibly inactivate C. albicans PMI. The addition of t he organic moiety increased the affinity of the compounds, with silver sulfadiazine showing a K-i of 190 +/- 30 nM. In all cases, the maximu m inhibition rate was similar, implying a similar rate-determining ste p. Silver sulfadiazine does not inhibit Escherichia coli PMI, and this suggests a role of the only free cysteine, Cys-150, in the inactivati on process. To confirm this, we mutated this residue to alanine in C. albicans PMI. The resultant Cys150 --> Ala mutant protein showed simil ar V-m and K-m values to the wild-type enzyme. However, it could not b e inhibited by silver sulfadiazine, and was 1000-fold less sensitive t o mercury inhibition. This confirms that Cys-150 is the site of action of the compounds in the C. albicans enzyme. All the silver-containing compounds inhibit human PMI with similar values for k(i)/K-i, in the range 0.43-1.67 mu M(-1). min(-1). By modifying the organic moiety, we have changed the compounds from a 210-fold bias toward inhibiting the mammalian enzyme to only 1.3-fold. The region surrounding Cys-150 pre sumably contains residues which could be important in the design of se lective PMI inhibitors.