Ternary products, direct formation from

Similar studies of the enzyme from pig skeletal muscle have been reported 175,183). In the earlier work, a fast burst of NADH formation in the dead-time of the apparatus was observed, equal in amplitude to the active center concentration at pH 8.0, but smaller at lower pH values. The suggestion that slow isomerization of the ternary product complex before pyruvate release may be the step responsible for the low steady-state maximum rate of lactate oxidation seems to be inconsistent with the full burst observed at pH 8.0, since it might be expected to result in partial equilibration of the reactant and product ternary complexes. Direct studies of the oxidation of E-NADH by pyruvate at pH 9.0 did indicate that reverse hydride transfer from NADH to pyruvate is indeed fast, but the absence of a deuterium isotope effect suggested that the observed rate constant of 246 sec, equal to the maximum steady-state rate of pyruvate reduction, may reflect an isomerization of the ternary complex preceding even faster hydride transfer. More recent studies 183) with improved techniques, however, appear to indicate no burst of enzyme-bound NADH formation preceding the steady-state phase of lactate oxidation at pH 8.0. On the basis of stopped-flow studies of lactate oxidation in the presence of oxamate, which forms a dead-end complex with E-NADH and can serve as an indicator of the rate of formation... 

Aldehyde formation during the catalytic action of the enzyme requires a net removal of two hydrogen atoms from the alcohol substrate. This dehydrogenation process is known to proceed by a mechanism of combined proton and hydride ion transfer, and it has been well established that transfer of hydride ion occurs directly between substrate and coenzyme in the productive ternary complex. 

Predominant P—O Fission. In the absence of Zn2+ ion, the reactions of PPS and PCA were very slow. Therefore, Zn2+ ion is essential for faster reaction. The kinetics described later indicate that the reaction proceeds through the formation of ternary complex (A) as illustrated in Figure 13. The oximate anion in A may either attack phosphorus (Path a) or sulfur (Path b). Inorganic sulfate was obtained quantitatively. This itself is not proof of Path a, because C (prepared separately) was found to be hydrolyzed readily to give sulfate under the same reaction conditions. However, the other isolated major product was B instead of the oxime catalyst that would be regenerated from C. The product B gave methylphenylphosphate when solvolyzed in methanol in the presence of Zn2+ ion. Methylphenylphosphate also was obtained directly from A in the reaction in methanol, whereas the formation of methylsulfate was not detected. Thus, these results all indicate that the Zn2+PCA complex promotes predominant P—O fission. 

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