The effect of amphipaths on the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from Escherichia coli.
When assayed by the phenazine methosulphate coupled 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from Escherichia coli strain 7 showed a marked loss of activity if depleted of detergent, and this activity could be reconstituted with amphipaths. However, 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 Michaelis-Menten constants for DL-glycerol 3-phosphate, as measured by the phenazine methosulphate coupled MTT and ferricyanide reduction assays in the presence of amphipath, were found to be 1.9 and 10.2 mM, respectively. Cupric ions were found to specifically inhibit the phenazine methosulphate coupled MTT reducing activity while zinc ions inhibited the ferricyanide reducing activity. Circular dichroic studies provided initial evidence for a conformation 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. We conclude from these studies that amphipaths may specificity induce a phenazine methosulphate binding site thus permitting reduction of this electron acceptor.
D000332:Aerobiosis; D002942:Circular Dichroism; D004120:Dimethyl Suberimidate; D004926:Escherichia coli / Q000201:enzymology*; D005415:Flavins; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000494:pharmacology
D005415:Flavins; D013501:Surface-Active Agents; D004120:Dimethyl Suberimidate; D005993:Glycerolphosphate Dehydrogenase