The rate-determining step in P450C21-catalyzing reactions in a membrane-reconstituted system

Citation
S. Kominami et al., The rate-determining step in P450C21-catalyzing reactions in a membrane-reconstituted system, J BIOL CHEM, 276(14), 2001, pp. 10753-10758
Citations number
43
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
0021-9258 → ACNP
Volume
276
Issue
14
Year of publication
2001
Pages
10753 - 10758
Database
ISI
SICI code
0021-9258(20010406)276:14<10753:TRSIPR>2.0.ZU;2-G
Abstract
Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21 -hydroxylation of 17 alpha -hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37 degreesC, The rate of product fo rmation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume o f the lipid phase. The rates of conversion of progesterone and 17 alpha -hy droxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 +/- 0.7 s( -1), and that for 17 alpha -hydroxyprogesterone was 1.8 +/- 0.5 s(-1) at 37 degreesC, It was found from the analysis of kinetic data that the rate-det ermining step in 21-hydroxylation of progesterone in the steady state was t he dissociation of product from P450 C21, whereas the conversion to deoxyco rtisol was the rate-determining step in the reaction of 17 alpha -hydroxypr ogesterone. The difference in the rate-determining steps in the reactions f or the two substrates was clearly demonstrated in the pre-steady-state kine tics.