Primary and secondary tritium kinetic isotope effects have been determined for the reactions catalyzed by purified preparations of γ-butyrobetaine hydroxylase obtained from Pseudomonas sp AK 1 and from calf liver. With [methyl-14C,(3R)-3-3H]-γ-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 γ-butyrobetaine. Under identical conditions of analysis, the calf liver enzyme exhibited a primary T(V/K) of ∼15. With [methyl-14C,(4R)-4-3H]-γ-butyrobetaine as substrate, a β-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 α-secondary T(V/K), as derived from measurements with [methyl-14C,2,3-3H]-γ-butyrobetaine, suggests that the motions of the primary and α-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.
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