A microcompartmentation analysis of intermediate leakage response to substrate excess in a membrane-bound bifunctional enzyme: local control of hydroxyprogesterone channeling efficiency during cytochrome P450XVII-catalysed androgen biosynthesis.
Evidence is presented for the first time that the cytochrome P450XVII-catalysed androgen formation from progesterone (P) in rat testicular microsomal membranes represents a metabolic sequence that exhibits the ability of intrinsic regulation of intermediate transfer and product formation efficiency. Exposure of this system, which catalyses a hydroxylation and oxidative cleavage reaction sequence, to increasing P concentration results in a decreased specific retention of the putative intermediate, 17 alpha-hydroxyprogesterone (HP) in the membrane compartment, and in a decreased HP conversion to androgens in favour of increasing HP transfer into the extramembrane space. This behaviour results in a decreased ratio of product vs. intermediate formation rates, which is interpreted as a partial "uncoupling" of the normal hydroxylation and cleavage reaction sequence catalysed by P450XVII. A similar pattern can likewise be observed in isolated testicular Leydig cells after exposure to increasing P concentrations under more physiological continuous-flow conditions. Further calculations indirectly indicate that the specific retention of HP in the membrane compartment can partially be attributed to its specific association with the P450XVII during catalysis. The results strongly suggest the existence of a local "channel" that becomes more leaky and therefore less effective if loaded with high influx rates. This pattern may be related to significant but incomplete competition of exogenously entering P and endogenously formed and transiently bound HP for oxygen attack at the P450XVII active site.
Kühn-Velten
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Institut für Physiologische Chemie II, Heinrich-Heine-Universität, Düsseldorf, Germany.
D000728:Androgens / Q000096:biosynthesis*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000494:pharmacology*; D006900:Hydroxylation; D006908:Hydroxyprogesterones / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D007985:Leydig Cells; D008297:Male; D008861:Microsomes / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology / Q000378:metabolism*
D000728:Androgens; D006908:Hydroxyprogesterones; D003577:Cytochrome P-450 Enzyme System