Abstract:
:The kinetic mechanism of NADPH-cytochrome P450 oxidoreductase (P450R) has been determined for the model reaction with cytochrome c3+. Although initial velocity studies show parallel patterns, consistent with a classical (one-site) ping-pong mechanism that precludes the formation of a ternary NADPH-P450R-cytochrome c3+ complex, product and dead-end inhibition results suggest a nonclassical (two-site) ping-pong mechanism [Northrop, D. B. (1969) J. Biol. Chem. 244, 5808-5819]. This mechanism is a hybrid of the random sequential (ternary complex) and ping-pong mechanisms, since ternary complexes can form as well as intermediate, modified forms of the enzyme that can be present in the absence of any bound substrate. The complete rate equation is derived for this mechanism, and values for Vmax, (V/K)NADPH, (V/K)cytc, and the corresponding Michaelis constants are presented in terms of microscopic rate constants along with the expected product inhibition patterns (Appendix). Inhibition by NADP+ is competitive versus NADPH and uncompetitive versus cytochrome c3+, while inhibition by cytochrome c2+ is competitive versus cytochrome c3+ and noncompetitive versus NADPH. These inhibition patterns are consistent with the proposed two-site mechanism. This mechanism would give the same initial velocity patterns as the classical one-site ping-pong mechanism, but it allows for the formation of a ternary complex, with NADPH and cytochrome c3+ reacting independently at two separate sites on P450R. The D(V/K)NADPH isotope effect is not affected by cytochrome c3+ concentration, consistent with our assumption (in deriving the rate equation) that binding at the two sites is independent. At the high ionic strength used in this study (850 mM), the mechanism is two-site ping-pong, with the electron acceptor site itself reacting with cytochrome c3+ in a tetra uni ping-pong manner.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Sem DS,Kasper CBdoi
10.1021/bi00206a002subject
Has Abstractpub_date
1994-10-11 00:00:00pages
12012-21issue
40eissn
0006-2960issn
1520-4995journal_volume
33pub_type
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