J. Biol. Chem., Vol. 259, Issue 11, 6748-6751, 06, 1984
Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. pH dependence of NADPH binding and isotope rate effect for beta-ketoacyl reductase
Z Yuan and GG Hammes
The stopped flow method has been used to determine the pH dependence of the
kinetics of the binding of NADPH to chicken liver fatty acid synthase over
the pH range 6.0-8.5. The kinetics is consistent with a one-step binding
mechanism, and the pH dependence of the second order rate constant
indicates that an ionizable group either on the enzyme or on NADPH with a
pK alpha of 6.1 is of importance in the binding process. The isotope rate
effects have been determined for the steady state reaction with (S)- and
(R)-[4-2H] NADPH as substrates and are very small. The pH dependence of the
rate constant characterizing the reduction of acetoacetyl by NADPH on the
enzyme (beta-ketoacyl reductase) and the isotope rate effects on this
constant with (S)-[4- 2H]NADPH as substrate also have been measured with
the stopped flow method. A small pH-dependent isotope rate effect is found;
these results suggest hydride transfer is not rate limiting for the beta-
ketoacyl reductase reaction on the enzyme surface. The pH dependence of
this rate constant is bell shaped and is very similar to that of the
turnover number for the overall reaction; this suggests that the beta-
ketoacyl reductase reaction may be partially rate limiting for the overall
reaction when the enzyme is saturated with substrates.
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