γ-Butyrobetaine Hydroxylase: Primary and Secondary Tritium Kinetic Isotope Effects

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.