title: Kinetic mechanism of lecithin retinol acyl transferase.
abstract: Lecithin retinol acyl transferase transfers acyl groups regiospecifically from the 1-position of lecithins to all-trans-retinol (vitamin A) and similar retinoids. LRAT is essential for the biosynthesis of 11-cis-retinal, the visual pigment chromophore, and is also required for the general dietary mobilization of vitamin A. The kinetic mechanism of this enzyme is described here, KM and Vmax values were determined for the substrates dipalmitoylphosphatidylcholine (DPPC) [1.38 microM and 0.17 microM/(min-mg), respectively] and for all-trans-retinol [0.243 microM and 0.199 microM/(min-mg), respectively]. In order to distinguish between a ping-pong bi-bi mechanism and a rapid equilibrium random or ordered bi-bi mechanism, the velocity of product formation as a function of one of the substrates at different fixed concentrations of the other substrate was measured. The parallel lines generated are entirely consistent with a ping-pong bi-bi mechanism in which DPPC first binds to LRAT and acylates it and rule out both simple random binding and ordered kinetic mechanisms. Further evidence for a ping-pong bi-bi mechanism comes from partial exchange reaction studies which show that LRAT can catalyze acyl group interchange between two different lecithin derivatives. Finally, the ping-pong reaction was established as being ordered, using the potent and reversible dead-end inhibitor 13-desmethyl-13,14-dihydro-all-trans-retinyl trifluoroacetate. This compound proved to be competitive with respect to DPPC, with a KI = 11.4 microM, and uncompetitive with respect to all-trans-retinol.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Shi
authors.forename: I
authors.initials: I
authors.lastname: Hubacek
authors.forename: R R
authors.initials: RR
authors.lastname: Rando
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D007202:Indicators and Reagents; D007700:Kinetics; D008962:Models, Theoretical; D010713:Phosphatidylcholines / Q000138:chemical synthesis* / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D012176:Retinoids / Q000138:chemical synthesis* / Q000378:metabolism; D013379:Substrate Specificity; D014801:Vitamin A / Q000378:metabolism
chemical_list: D007202:Indicators and Reagents; D010713:Phosphatidylcholines; D012176:Retinoids; D014801:Vitamin A; D015060:1,2-Dipalmitoylphosphatidylcholine; D000217:Acyltransferases; C056942:lecithin-retinol acyltransferase