Proline dehydrogenase from Escherichia coli K12. Properties of the membrane-associated enzyme.
We have examined the oxidative activities of inverted cytoplasmic membrane preparations from Escherichia coli bearing proline dehydrogenase. Our measurements include both direct substrate:2,6-dichloroindophenol and substrate:O2 oxidoreductase assays and the 9-aminoacridine fluorescence assay for proton translocation, employing succinate and NADH dehydrogenases as comparative standards. Our data show the following. (a) Membranes prepared in a new buffer system bear proline dehydrogenase that is stable in both activity and membrane association. This membrane-associated enzyme shows an apparent Km for proline 20-fold lower than that estimated from the solubilized and purified enzyme. (b) Electrons are transferred from proline to O2 via the respiratory chain since proline utilization requires porphyrin synthesis and it is coupled to trans-membrane proton translocation. (c) Patterns of inhibition by 5-ethyl-5-isopentyl barbituric acid (Amytal) and 2-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO) suggest that parallel pathways of electron flux from NADH and proline converge at a cyanide-sensitive terminal oxidase. Succinate:O2 and succinate:DCIP oxidoreductase activities are stimulated by HpHOQnO and Amytal, and the former is inhibited by cyanide in this system. (d) Amytal is a non-competitive inhibitor of proline dehydrogenase. (e) Analysis of our fluorescence data suggests that Amytal and HpHOQnO dissipate delta pH at concentrations as low as 5 mM and 8.5 microM, respectively, in this system.
D002462:Cell Membrane / Q000201:enzymology; D004579:Electron Transport / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification*; D011394:Proline Oxidase / Q000302:isolation & purification*; D011522:Protons; D013050:Spectrometry, Fluorescence
D011522:Protons; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011394:Proline Oxidase