Kinetic analysis of mouse retinal dehydrogenase type-2 (RALDH2) for retinal substrates

Isabelle Gagnon , Gregg Duester , Pangala V. Bhat
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引用次数: 66

Abstract

Retinal dehydrogenase (RALDH) isozymes catalyze the terminal oxidation of retinol into retinoic acid (RA) that is essential for embryogenesis and tissue differentiation. To understand the role of mouse type 2 RALDH in synthesizing the ligands (all-trans and 9-cis RA) needed to bind and activate nuclear RA receptors, we determined the detailed kinetic properties of RALDH2 for various retinal substrates. Purified recombinant RALDH2 showed a pH optimum of 9.0 for all-trans retinal oxidation. The activity of the enzyme was lower at 37°C compared to 25°C. The efficiency of conversion of all-trans retinal to RA was 2- and 5-fold higher than 13-cis and 9-cis retinal, respectively. The Km for all-trans and 13-cis retinal were similar (0.66 and 0.62 μM, respectively). However, the Km of RALDH2 for 9-cis retinal substrate (2.25 μM) was 3-fold higher compared to all-trans and 13-cis retinal substrates. Among several reagents tested for their ability to either inhibit or activate RALDH2, citral and para-hydroxymercuribenzoic acid (p-HMB) inhibited and MgCl2 activated the reaction. Comparison of the kinetic properties of RALDH2 for retinal substrates and its activity towards various reagents with those of previously reported rat kidney RALDH1 and human liver aldehyde dehydrogenase-1 showed distinct differences. Since RALDH2 has low Km and high catalytic efficiency for all-trans retinal, it may likely be involved in the production of all-trans RA in vivo.

小鼠视网膜底物脱氢酶2型(RALDH2)的动力学分析
视网膜脱氢酶(RALDH)同工酶催化视黄醇最终氧化为视黄酸(RA),这是胚胎发生和组织分化所必需的。为了了解小鼠2型RALDH在合成结合和激活核RA受体所需的配体(全反式和9顺式RA)中的作用,我们确定了RALDH2对各种视网膜底物的详细动力学性质。纯化的重组RALDH2在全反式视网膜氧化中pH值为9.0。与25℃相比,37℃时酶的活性较低。全反式视网膜转化为RA的效率分别比13-顺式和9-顺式视网膜高2倍和5倍。全反式和13顺式视网膜的Km值相近,分别为0.66和0.62 μM。然而,9-顺式视网膜底物的RALDH2的Km (2.25 μM)比全反式和13-顺式视网膜底物高3倍。在几种抑制或激活RALDH2的试剂中,柠檬醛和对羟基氨基苯甲酸(p-HMB)抑制了该反应,而MgCl2则激活了该反应。RALDH2对视网膜底物的动力学性质及其对各种试剂的活性与先前报道的大鼠肾脏RALDH1和人肝脏醛脱氢酶-1的动力学性质比较显示出明显的差异。由于RALDH2对全反式视网膜具有较低的Km和较高的催化效率,在体内可能参与了全反式RA的生成。
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