The stereochemical course of the aliphatic hydroxylation of γ-butyrobetaine by calf liver and by Pseudomonas sp AK1 γ-butyrobetaine hydroxylases has been determined. With [3(.RS)-3-3H]-γ-butyrobetaine or [3(R)-3-3H]-γ-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-γ-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(5)-3-2H]-γ-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product l-carnitine was found to have retained all of the deuterium initially present in the 3(5) position. Since the absolute configuration of the product l-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [metheyl-14C,3(R)-3-3H]-γ-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [metheyl-14C]-l-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [metheyl-I4C,3(RS)-3-3H]-γ-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate α-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for γ-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.
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