The effect of amphipaths on the flavin-linked aerobic glycerol-3-pliosphate dehydrogenase from Escherichia coli

Abstract

When assayed by the phenazine methosulfate coupled 3-(4,5-dimethylthylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from E. coli strain 7 showed a marked loss of activity if depleted of detergent, and this activity could be reconstituted with amphipaths. Enzyme activity, as measured by the ferricyanide reduction assay, showed no loss of activity upon detergent depletion. Cross-linking studies indicated that amphipath was not inducing any significant quaternary structural change in the enzyme. Flavin fluorescence titration experiments indicated a single type of binding site for glycerol 3-phosphate whose affinity was unaffected by the presence of amphipath; apparent Kd values of 19.5 and 17.1 mM were measured in the presence and absence of amphipath, respectively. The Km for DL-glycerol 3-phosphate, as measured by the phenazine methosulphate coupled MTT and ferricyanide reduction assays in the presence of amphipath, were 1.9 and 10.2 mM, respectively. Cupric ions specifically inhibited the phenazine methosulfate coupled MTT reducing activity while zinc ions inhibited the ferricyanide reducing activity. Circular dichroic studies provided initial evidence for a conformational change in the presence of the nonionic detergent Brij 58 and this was substantiated by quenching of tryptophan fluorescence at concentrations of Brij 58 equivalent to those needed to stimulate activity. Amphipaths may specifically induce a phenazine methosulfate binding site thus permitting reduction of this electron acceptor.