49 research outputs found
Protective effects of ibuprofen and its major metabolites against in vitro inactivation of catalase and fumarase: relevance to cataract
A transition from ionic to free-radical mechanisms in chemistry and enzymology
Abstract: 1) Examples were presented of enzymes which catalyse different generic reactions at the same These enzymes which belong to the P-450 class are aromatase, 1401-active site. demethylase, 17a-hydroxylase-17,20-lyase and nitric oxide synthase. 2) The multicatalysis is due to a selection process in which the functional group at the sensitive C-atom of the substrate chooses a compatible iron-oxygen species (Ferv0. or Fe"'-OOH) for further reaction. 3) The P-450 dependent hydroxylation and C-C bond cleavage reactions occur via a radical mechanism and the enzymes participating in these processes have evolved to deal with situations where the ionic processes are deemed energetically unfavourable. In this lecture, attention was focused on the transition from heterolytic to homolytic catalysis in chemical and enzymic reactions. It was argued that for C-H and C-C bond cleavage to occur via a carbanion the pKa of its conjugate acid may not be greater that 25; beyond this value free radical mechanisms are chosen in chemistry as well as enzymology for review see 1). The latter 2 is produced by a two-electron reduction of 0, as shown in Scheme 1. Although several electronically equivalent structures for the species are possible, the formulation Fe"-0' 2C, which bears an uncanny resemblance to the chemists' alkoxy radical, can be most conveniently used to describe the hydroxylation reaction in a step-wise fashion (4). In our laboratory, studies on aromatase (5) that is involved in the aromatisation of androgens and 14a-demethylase (6) that converts lanosterol into sterols revealed several new facets of P-450 dependent enzymes. These two multifunctional P-450 enzymes are involved not only in the hydroxylation reactions but also in the oxidation of alcohols into carbonyl compounds as well as in the cleavage of C-C bonds by an acyl-carbon fission as shown below
Mechanistic kinship between hydroxylation and desaturation reactions: acyl-carbon bond cleavage promoted by pig and human CYP17 (P-45017.alpha.; 17.alpha.-hydroxylase-17,20-lyase)
The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14 alpha-demethylase of Candida albicans (other names are: lanosterol 14 alpha-demethylase, P-45014DM, and CYP51)
The Candida albicans sterol 14 alpha-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 mu mol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14 alpha-demethylase, 3 beta-hydroxylanost-7-en-32-al and 3 beta-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3 beta-hydroxylanost-7-en-32-al with the recombinant 14 alpha-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17 alpha-hydroxylase-17,20-lyase (P-45017 alpha,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14 alpha-demethylase is considered
The mutation t315a in candida albicans sterol 14 alpha demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity
Sterol 14alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity