From a study of the steady-state kinetics (at pH 7.6, 30 °C) of the reduction of cytochrome c, a ‘ping-pong’ mechanism may be postulated for the crystalline NADPH-cytochrome c reductase from ale yeast, Saccharomyces cerevisiae [1], a result derivable from a three-substrate ordered system with a rapid equilibrium random sequence in substrates,NADPH and FAD, followed by reactions of the third substrate, Cyt C^3+. On this basis, estimates for the kinetic parameters were made together with the inhibitor dissociation constants for NADP^+ (competitive with respect to NADPH as variable substrate, but noncompetitive with respect to cytochrome c^3+ as the variable substrate). A noncompetitive type of inhibition was also found for cytochrome c^2+ with NADPH as variable substrate, in confirmation of the proposed mechanism. With 2,6-dichloroindophenol as the acceptor, in place of cytochrome c^3+,a value for K(NADPH) could be estimated which agreed with that estimated above, with cytochrome c^3+ as the acceptor, again, in confirmation of the postulated mechanism. The reactions with molecular 0(2) catalyzed by the enzyme with NADPH as the reductant have been studied polarographically, and its K(m) for 0(2) estimated to be about 0.15 mmol/1 at pH 7.6, 25 °C. The product of the reaction appears to be H(2)0(2), which acts as a noncompetitive inhibitor for NADPH (K(1) = 0.5 mmol/1), and tentatively an enzyme ternary complex containing oxygen and FAD(h) (semiquinone of FAD) may be assumed to be the kinetically important intermediate, which may be postulated to be in quasi-equilibrium with an enzyme ternary complex containing 02 (superoxide) and FAD.

Keywords:
NADPH-cytochrome c reductase, Steady-state kinetics, Ale yeast enzyme, Reductase, Enzyme mechanism
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1984
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