Kinetic isotope effects enzymic bond cleavage

The physiological pathway of electron transfer in flavocytochrome is from bound lactate to FMN, then FMN to 52-heme, and finally 52-heme to cytochrome c (Fig. 9) (2,11, 80,102). The first step, oxidation of L-lactate to pyruvate with concomitant electron transfer to FMN, is the slowest step in the enzyme turnover (103). With the enzyme from S. cerevisiae, a steady-state kinetic isotope effect (with ferricyanide as electron acceptor) of around 5 was obtained for the oxidation of dl-lactate deuterated at the C position, consistent with the major ratedetermining step being cleavage of the C -H bond (103). Flavocytochrome 52 reduction by [2- H]lactate measured by stopped-flow spectrophotometry resulted in isotope effects of 8 and 6 for flavin and heme reduction, respectively, indicating that C -H bond cleavage is not totally rate limiting (104). 

Table 5.2 VjK) kinetic isotope effects on enzymic nucleoside bond cleavage (intrinsic-position is given.

The key experiment in relating the mechanism of hypophosphite inactivation to the normal PFL reaction is the observation of a primary kinetic isotope effect on the inactivation process with [ H2]hypophosphite 187). An isotope effect of 2.6 is reported with the free enzyme (0.25 mM hypophosphite), and a smaller but still primary effect of 1.6 for the acetyl-enzyme. This suggests that phosphoms-hydrogen bond cleavage is at least partially rate limiting during inactivation. These studies have led Kozarich and co-workers to propose a preliminary mech-... 

The means by which enzymes facilitate the homolytic cleavage of the Co-C5 bond has been addressed in detail in studies of MCM. This process can be kinetically monitored by the spectral change from Co(III) in coenzyme B12 to that of cob(II)alamin. This spectral change depends on the addition of the substrate to the complex of MCM and adenosylcobalamin. The kinetic barrier to bond cleavage is lowered by 17 kcalmol . " " Moreover, the rate of this change displays a kinetic isotope effect of >20 (Fh/F ) when the deuterated substrate is employed. " It was concluded that Co-C5 bond cleavage and hydrogen abstraction from the substrate are kinetically coupled. This effect has been reported for other coenzyme Bj2-dependent reactions as well. 

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