Engel C K, Mathieu M, Zeelen J P, Hiltunen J K, Wierenga R K
European Molecular Biology Laboratory, Heidelberg, Germany.
EMBO J. 1996 Oct 1;15(19):5135-45.
The crystal structure of rat liver mitochondrial enoyl-coenzyme A (CoA) hydratase complexed with the potent inhibitor acetoacetyl-CoA has been refined at 2.5 angstroms resolution. This enzyme catalyses the reversible addition of water to alpha,beta-unsaturated enoyl-CoA thioesters, with nearly diffusion-controlled reaction rates for the best substrates. Enoyl-CoA hydratase is a hexamer of six identical subunits of 161 kDa molecular mass for the complex. The hexamer is a dimer of trimers. The monomer is folded into a right-handed spiral of four turns, followed by two small domains which are involved in trimerization. Each turn of the spiral consists of two beta-strands and an alpha-helix. The mechanism for the hydratase/dehydratase reaction follows a syn-stereochemistry, a preference that is opposite to the nonenzymatic reaction. The active-site architecture agrees with this stereochemistry. It confirms the importance of Glu164 as the catalytic acid for providing the alpha-proton during the hydratase reaction. It also shows the importance of Glu144 as the catalytic base for the activation of a water molecule in the hydratase reaction. The comparison of an unliganded and a liganded active site within the same crystal form shows a water molecule in the unliganded subunit. This water molecule is bound between the two catalytic glutamates and could serve as the activated water during catalysis.
与强效抑制剂乙酰乙酰辅酶A复合的大鼠肝脏线粒体烯酰辅酶A水合酶的晶体结构已在2.5埃分辨率下得到优化。该酶催化水可逆地加成到α,β-不饱和烯酰辅酶A硫酯上，对于最佳底物，其反应速率接近扩散控制。烯酰辅酶A水合酶复合物是由六个相同的161 kDa分子质量亚基组成的六聚体。六聚体是三聚体的二聚体。单体折叠成一个四圈的右手螺旋，后面跟着两个参与三聚化的小结构域。螺旋的每一圈由两条β链和一个α螺旋组成。水合酶/脱水酶反应的机制遵循顺式立体化学，这一偏好与非酶促反应相反。活性位点结构与这种立体化学一致。它证实了Glu164作为催化酸在水合酶反应中提供α-质子的重要性。它还显示了Glu144作为催化碱在水合酶反应中激活水分子的重要性。同一晶体形式中未结合和结合的活性位点的比较显示，未结合亚基中有一个水分子。这个水分子结合在两个催化谷氨酸之间，可能在催化过程中作为被激活的水。
