Francesconi M A, Donella-Deana A, Furlanetto V, Cavallini L, Palatini P, Deana R
Department of Biological Chemistry, University of Padova, Italy.
Biochim Biophys Acta. 1989 Nov 30;999(2):163-70. doi: 10.1016/0167-4838(89)90213-6.
Succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase, previously identified in rat-liver mitochondria (Deana et al. (1981), Biochim. Biophys. Acta 662, 119-124), was purified to near homogeneity and further characterized. After the last purification steps consisting of Ultrogel AcA-44 filtration and agarose-hexane-coenzyme A chromatography, the enzyme was apparently tetrameric with a mass of 48-52 kDa determined by gel filtration on Sephadex G-75, ultracentrifugation through a sucrose gradient and SDS-gel electrophoresis. By means of a HPLC technique developed for measuring the CoA esters we could determine the enzyme activity in both forward and reverse directions and show that the kinetic constants, i.e., Km of reactants and Vmax, are not too different for the two reactions. Double-reciprocal plots of the enzyme velocities versus the concentration of one substrate at different fixed concentrations of the other substrate gave families of straight lines converging below the substrate-abscissa for both forward and backward reactions, indicating a kinetic mechanism of rapid equilibrium random Bi-Bi type. The competitive inhibition of the product succinate with respect to both reactants, 3-hydroxy-3-methylglutarate and succinyl-CoA, as well as the Haldane relationships are consistent with this conclusion. An inhibitory effect on CoA transferase activity by acetate, acetoacetate, acetyl-CoA, acetoacetyl-CoA, coenzyme A, carnitine, ZnCl2 and high concentrations of the monovalent anions ClO4-, F-, I- and Cl- was also found.
琥珀酰辅酶A：3-羟基-3-甲基戊二酸辅酶A转移酶，先前已在大鼠肝脏线粒体中鉴定出来（迪安娜等人，（1981年），《生物化学与生物物理学报》662卷，第119 - 124页），被纯化至接近均一状态并进一步进行了特性分析。在经过由Ultrogel AcA - 44过滤和琼脂糖 - 己烷 - 辅酶A层析组成的最后纯化步骤后，通过在Sephadex G - 75上进行凝胶过滤、通过蔗糖梯度进行超速离心以及SDS - 凝胶电泳测定，该酶显然为四聚体，质量为48 - 52 kDa。借助为测定辅酶A酯而开发的高效液相色谱技术，我们能够测定该酶正向和反向反应的活性，并表明这两个反应的动力学常数，即反应物的Km和Vmax，并无太大差异。在不同固定浓度的另一种底物存在下，酶速度与一种底物浓度的双倒数作图，对于正向和反向反应均给出了在底物横坐标下方收敛的直线族，表明其动力学机制为快速平衡随机双底物双产物类型。产物琥珀酸对反应物3 - 羟基 - 3 - 甲基戊二酸和琥珀酰辅酶A的竞争性抑制作用以及Haldane关系均与此结论相符。还发现乙酸、乙酰乙酸、乙酰辅酶A、乙酰乙酰辅酶A、辅酶A、肉碱、ZnCl2以及高浓度的单价阴离子ClO4 - 、F - 、I - 和Cl - 对辅酶A转移酶活性有抑制作用。
