Gagnon Isabelle, Duester Gregg, Bhat Pangala V
Laboratory of Nutrition and Cancer, Research Centre, Centre hospitalier de l'Universite de Montreal-Hotel-Dieu, 3850 St. Urbain Street, Montreal, Quebec, Canada H2W 1T7.
Biochem Pharmacol. 2003 May 15;65(10):1685-90. doi: 10.1016/s0006-2952(03)00150-3.
Retinal dehydrogenases (RALDHs) convert retinal into retinoic acids (RAs), which are important signaling molecules in embryogenesis and tissue differentiation. We expressed mouse RALDH type 1 (mRALDH1) in Escherichia coli and studied the kinetic properties of the recombinant enzyme for retinal substrates. Purified recombinant mRALDH1 catalyzed the oxidation of all-trans and 9-cis retinal but not 13-cis retinal, and exhibited two pH optimums, 7.8 and 9.4, for all-trans and 9-cis retinal substrates, respectively. The K(m) for all-trans retinal (11.6 micro M) was 3-fold higher than for 9-cis retinal (3.59 micro M). However, the conversion efficiencies of either all-trans or 9-cis retinal to the respective RAs were similar. MgCl(2) inhibited the oxidation of both all-trans and 9-cis retinal. Chloral hydrate and acetaldehyde competitively suppressed all-trans retinal oxidation with inhibition constants (K(i)) of 4.99 and 49.4 micro M, respectively. Retinol, on the other hand, blocked the reaction uncompetitively. These data extend the kinetic characterization of mRALDH1, provide insight into the possible role of this enzyme in the biogenesis of RAs, and should give useful information on the determination of amino acid residues that play crucial roles in the catalysis of all-trans and 9-cis retinal.
视网膜脱氢酶（RALDHs）将视黄醛转化为视黄酸（RAs），视黄酸是胚胎发育和组织分化过程中重要的信号分子。我们在大肠杆菌中表达了小鼠1型RALDH（mRALDH1），并研究了该重组酶对视黄醛底物的动力学特性。纯化的重组mRALDH1催化全反式视黄醛和9-顺式视黄醛的氧化，但不催化13-顺式视黄醛的氧化，并且分别对全反式视黄醛和9-顺式视黄醛底物表现出7.8和9.4两个最适pH值。全反式视黄醛的米氏常数（K(m)）（11.6微摩尔）比9-顺式视黄醛（3.59微摩尔）高3倍。然而，全反式视黄醛或9-顺式视黄醛转化为各自视黄酸的效率相似。MgCl₂抑制全反式视黄醛和9-顺式视黄醛的氧化。水合氯醛和乙醛竞争性抑制全反式视黄醛的氧化，抑制常数（K(i)）分别为4.99和49.4微摩尔。另一方面，视黄醇非竞争性地阻断反应。这些数据扩展了mRALDH1的动力学特征，深入了解了该酶在视黄酸生物合成中的可能作用，并应为确定在全反式视黄醛和9-顺式视黄醛催化中起关键作用的氨基酸残基提供有用信息。
