Glyceryl-ether monooxygenase [EC 1.14.16.5]. Part 9. Stereospecificity of the oxygenase reaction

Abstract

(2RS,1′R)-[1′-³H₁ ]- and (2RS, 1′S)-[1′-³H₁]-Hexade cyloxypropane-1,2-diols (chimyl alcohols) have been prepared and their stereochemistry has been confirmed by synthesizing the [²H₁]-analogues using similar procedures. When they were used as substrates for glyceryl-ether monooxygenase from rat liver in the presence of oxygen and (RS)-6-methyl-5,6,7,8-tetrahydropterin as co-factor, the 1′S-isomer released 37% of its tritium into the aqueous buffer after 20 mins, whereas the 1′R-isomer released only 6.5% showing that the reaction was stereospecific for the pro-H_S hydrogen atom of the glyceryl ether substrate. This was in agreement with the kinetic parameters of unlabelled-(2RS)-3-, (2RS, 1′R)-3-[1′-²H₁]-, (2RS, 1′S)-3-[1′-²H₁]- and (2RS)-3-[1′,1′-²H₂]- hexadecyloxypropane-1,2-diols where the apparent K_m values were about the same (49.4, 53.7, 49.3 and 54.0 µM respectively) but the apparent maximum velocities (V_max in nmol min^-1 mg^-1 protein) of the first two substrates (37.5 and 37.5) were faster than for the latter two substrates (22.5 and 23.6), consistent with the pro-H_S hydrogen atom being replaced by the hydroxy group and a primary deuterium isotope effect of ≈1.6.