Chicken Liver Fatty Acid Synthase pH DEPENDENCE OF NADPH BINDING AND ISOTOPE RATE EFFECT

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, of 6.1 is of importance in the binding process. The isotope rate effects have been determined for the steady state reaction with (27)and (B)-[4-2H] NADPH as substrates and are very small. The pH dependence of the rate constant characterizing the r duction of acetoacetyl by NADPH on the enzyme (& 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 ffect is found; these results suggest hydride transfer is not rate limiting for the 8ketoacyl 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 @-ketoacyl reductase reaction may be partially rate limiting for the overall reaction when the enzyme is saturated with substrates.