Inhibition of aldosterone production in rat adrenal mitochondria by 18-ethynyl-11-deoxycorticosterone: a simple model for kinetic interpretation of mechanism-based inhibitors.
A simple mathematical model for studying mechanism-based inhibitors (MBIs) is presented. The mathematical equations are deduced for an experimental protocol consisting of a first incubation of the enzyme in the presence of MBI followed by a washing protocol to eliminate free MBI. Finally enzyme activity (initial velocity) is measured with specific substrate. The representation of the final equation obtained is a straight line, and the MBI-specific association constant of velocity (k) can be calculated from its slope. The mathematical model was then challenged with the effect of 18-ethynyl-11-deoxycorticosterone (18-EtDOC) as an MBI on aldosterone biosynthesis from 11-deoxycorticosterone (DOC) in rat adrenal mitochondria. The last step of the mitochondrial biosynthesis of aldosterone consists of the conversion of DOC into corticosterone (B) or 18-hydroxy-11-deoxycorticosterone (18-OHDOC), and both steroids can then be transformed into aldosterone. The k (mM(-1) x min(-1)) values obtained for 18-EtDOC were: 451 +/- 36 for DOC to aldosterone; 177 +/- 16 for B to aldosterone; 175 +/- 15 for 18-OHDOC to aldosterone; and 2.7 +/- 0.2 for DOC to B. These results show that this MBI practically does not affect the metabolism of DOC to B in our enzyme preparation and that conversions of B and 18-OHDOC into aldosterone are catalyzed by the same enzyme.
Matković
L
L
Department of Biological Chemistry, Faculty of Sciences, University of Buenos Aires and PRHOM-CONICET, 1428-Buenos Aires, Argentina. lmat@qb.fcen.uba.ar
Gomez-Sanchez
C E
CE
Cozza
E N
EN
D000302:Adrenal Cortex / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D000450:Aldosterone / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003345:Corticosterone / Q000378:metabolism; D003900:Desoxycorticosterone / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008954:Models, Biological; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D015384:Zona Glomerulosa / Q000166:cytology / Q000201:enzymology / Q000378:metabolism
D004791:Enzyme Inhibitors; D003900:Desoxycorticosterone; D000450:Aldosterone; D009249:NADP; C071418:18-ethynyldeoxycorticosterone; D002786:Cholesterol Side-Chain Cleavage Enzyme; D003345:Corticosterone