A Convenient Spectrophotometric Method for Measuring the Kinetic Parameters of Glyceryl-Ether Monooxygenase (EC 1.14.16.5)

Glyceryl-ether Monooxygenase [EC 1.14.16.5J Part V: Some Aspects of the Stoichiometry

“…The molar amounts of 6-MeP~ oxidised during the first two minutes were equal to the molar amounts of batyl alcohol consumed. This validates all the measurements of the kinetic parameters of the monooxygenase with lipid ether substrates and tetrahydropterin cofactors using the direct assay (8,9,18) because all the data were evaluated from initial rate kinetics measured within the first two minutes of reaction. Since the amount of tetrahydropterin oxidised is measured by the rate of q-6-MePH2 (4) produced, then the formation of a 4a-hydroxypterin intermediate (5) is not rate limiting during the first two minutes (see below).…”

“…Measurements of the kinetic parameters (Km and V max) of R-(natural)*, S-and RS-batyl alcohol, and of S-(natural)*-and RS-(racemic)-3-(1-hexadecyloxy)-2-hydroxypropane-l-phosphocholine (ryso-PAF) showed that the natural isomers in these ethers were marginally more active substrates than their enantiomers and racemates (9). Racemic batyl alcohol has therefore been used in all the present work.…”

“…After ca 8 minutes of reaction oxidation of 6-MePH4 appeared to stop but batyl alcohol continued to be hydroxylated, as observed by decrease in its concentration and the formation of more aliphatic aldehyde. It is known that the microsomal preparation has DHPR activity (9), and trace amounts of NADH (in the presence of other oxidoreductases and aliphatic aldehyde) may well assist in regenerating some ,6-MePH 4 (and keep its concentration steady at this stage of the reaction) while lipid ether is hydroxylated further (see amounts formed after 30 minutes, Figure I).…”

“…This was attributed to the fact that the pterin used was a DL mixture and that only 50% was enzymically active. However, the similarity of the apparent Vmax values of RS-6-methyl-, RS-6,7-dimethyl-5,6,7,8-tetrahydropterin and natural R-5,6,7,8-tetrahydrobiopterin (9) suggests that there is no stereoselectivity by the monooxygenase for the pterin cofactor. It was also found (6) that in a coupled system (Scheme 1), the amount of NADH oxidised per minute was equivalent to the total amount of fatty aldehyde, fatty alcohol and fatty acid produced.…”

“…We were interested in the stoichiometry of the reaction because very little was known about it and because we can now follow monooxygenase activity with the simpler direct spectrophotometric assay that we developed (9) in which the activity can be measured by the rate of oxidation of 5,6,7,8-tetrahydropterins (e.g. 3) to quinonoid-7,8(6 H)-dihydropterins (e.g.…”