Light-induced activation and synchronization of the cytochrome P-450 dependent monooxygenase system.
The light-induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system, consisting of the enzyme P-450PB-B and the NADPH-cytochrome P-450 reductase. The relative increase of the activity was about 15%. It is shown that the product release process is accelerated by light. The phases of the catalytic cycle of 2 x 10(12) protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.32 s repetition time, and 390-470 nm, 0.27 joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes (= "molecular machines") worked in phase after a few light pulses. The phase relation continued even after switching off the light pulses. The catalytic cycle time was 1.54 s, giving a turnover number of 39 min-1. The turnover number, as determined from the enzyme activity under optimum conditions, was 39 min-1. Due to the dissociation constant of the P-450PB-B:NADPH-P-450 reductase complexes [3] only 24% of the proteins were in the active (= working) state under the conditions used. The lifetime of this complexes is larger than 6 s since more than 4 cycles of the free running enzyme can be observed. This is the first report, that all catalytic active complexes in the test tube can be synchronized by an external light source, if the right repetition time of the pulses is chosen, so that all these "molecular machines" work in phase.
Häberle
W
W
Abteilung Biophysik, Universität Ulm, Bundesrepublik Deutschland.
Gruler
H
H
Dutkowski
P
P
Müller-Enoch
D
D
D015660:7-Alkoxycoumarin O-Dealkylase / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D000818:Animals; D003574:Cytochrome c Group / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008027:Light; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010634:Phenobarbital; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
D003574:Cytochrome c Group; D015660:7-Alkoxycoumarin O-Dealkylase; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital