.gamma.-Butyrobetaine hydroxylase: primary and secondary tritium kinetic isotope effects

Abstract

Primary and secondary tritium kinetic isotope effects have been determined for the reactions catalyzed by purified preparations of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp AK 1 and from calf liver. With [methyl-14C,(3R)-3-3H]-gamma-butyrobetaine as substrate, the bacterial hydroxylase was found to exhibit a primary T(V/K) of 1.3-1.5. This value was determined from measurements of either the specific activity of the medium 3H2O or of the ratio of 3H/14C in the residual gamma-butyrobetaine. Under identical conditions of analysis, the calf liver enzyme exhibited a primary T(V/K) of approximately 15. With [methyl-14C,(4R)-4-3H] gamma-butyrobetaine as substrate, a beta-secondary T(V/K) of 1.10 has been determined for the calf liver hydroxylase; this supports the existence in the reaction mechanism of an sp2-hybridized transition state. A large normal value of 1.31 for the alpha-secondary T(V/K), as derived from measurements with [methyl-14C,2,3-3H]-gamma-butyrobetaine, suggests that the motions of the primary and alpha-secondary hydrogens are coupled in the C-H cleavage step and resulting synchronous rehybridization. A chemical mechanism involving homolytic cleavage of the C-H bond at the position undergoing hydroxylation is proposed and discussed.

Isolation and Biochemical Characterization of Hypophosphite/ 2-Oxoglutarate Dioxygenase

“…A similar assay without HtxA was used as the enzyme-free control. Both mixtures were incubated with constant mixing at room temperature for about 2 h, after which samples were removed, D 2 O was added to 10% final concentration, and the samples were analyzed with 31 P NMR. The phosphite only standard contained 5 mM phosphite in 20 mM MOPS buffer.…”

“…Given the strict substrate specificity of PtxD determined in a previous study (15), it seems evident that phosphite is the phosphorus product of the HtxA reaction. However, to identify the product of the HtxA reaction unequivocally, 31 P NMR was used to analyze the reaction products (Fig. 4).…”

“…4. HtxA catalyzes the oxidation of hypophosphite to phosphite as detected by 31 P NMR. 31 P NMR was performed as described under "Experimental Procedures."…”

“…31 P NMR was performed as described under "Experimental Procedures." Each proton-coupled 31 P NMR spectrum shows the following: A, 5 mM phosphite standard showing the doublet peak from the phosphite signal; B, sample taken from the no enzyme control, showing the triplet peak of the hypophosphite signal; C, sample from the complete assay mixture containing 20 mM MOPS, pH 7.0, 5 mM hypophosphite, 3 mM 2-oxoglutarate, 100 M FeCl 2 , and 0.5 mg/ml HtxA, showing both the hypophosphite triplet and the phosphite doublet peaks. The vertical scale in each panel is adjusted for each spectra individually.…”

“…The commonality among all of the members of this family is that they all require activation of a molecule of dioxygen by enzyme-bound ferrous ions to generate a highly reactive ferryl oxidant. The formation of the ferryl species is linked to the oxidative decarboxylation of 2-oxoglutarate, giving rise to succinate and CO 2 , and mediates hydroxylation of the substrate (31,32). Thus, all members of this family are dependent on ferrous ions, oxygen, and 2-oxoglutarate (or a similar 2-oxoacid) for activity.…”