title: Proline dehydrogenase from Escherichia coli K12. Reconstitution of a functional membrane association.
abstract: Soluble and membrane associated proline dehydrogenase differ in catalytic properties. The soluble enzyme transfers electrons from L-proline to exogenous electron acceptors. It has a high Km for L-proline (105 mM) and is insensitive to the respiratory chain inhibitors 5-ethyl-5-isopentyl-barbituric acid and cyanide. The membrane-associated enzyme transfers electrons from L-proline to O2 via the respiratory chain, with coupled transmembrane proton translocation. It has a low Km for L-proline (3 mM) and is inhibited by 5-ethyl-5-isopentyl-barbituric acid and cyanide. Proline:O2 oxidoreductase activity identical to that of native membranes can be reconstituted using enzyme purified in the absence of detergent and enzyme deficient membranes from a putA mutant strain. Reassociation of the enzyme with the membrane is an autocatalytic process that requires the simultaneous presence of L-proline, MgCl2, enzyme, and membranes. It can be monitored by observing the chromogenic reaction of delta 1-pyrroline carboxylic acid with o-aminobenzaldehyde. Reduction of membrane components or generation of a protonmotive force is apparently required to promote enzyme-membrane association or to activate electron transfer. The reconstituted activity is a saturable function of enzyme concentration at constant membrane concentration and the activity approached is 20-fold higher than that of native membranes isolated from bacteria that have been induced for proline utilization. It is therefore unlikely that saturation of the available membrane binding sites is achieved during induction of the put genes in vivo.
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D011393:Procollagen-Proline Dioxygenase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011393:Procollagen-Proline Dioxygenase