Porter D J, Boyd F L
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1991 Nov 15;266(32):21616-25.
The kinetic mechanism of S-adenosylhomocysteine hydrolase was investigated by stopped-flow spectrofluorometry at pH 7.0 and 25 degrees C. Pre-steady-state kinetic steps were identified with chemical steps proposed for the mechanism of this enzyme (Palmer, J.L., and Abeles, R.H. (1979) J. Biol. Chem. 254, 1217-1226). The steady-state kinetic constants for the hydrolysis or synthesis of S-adenosylhomocysteine were in good agreement with those values calculated from the pre-steady-state rate constants. The equilibrium constant for dehydration of 3'-ketoadenosine to 3'-keto-4',5'-dehydroadenosine on the enzyme was 3. The analogous equilibrium constant for addition of L-homocysteine to S-3'-keto-4',5'-dehydroadenosylhomocysteine on the enzyme was 0.3. The elimination of H2O from adenosine in solution had an equilibrium constant of 1.4 (aH2O = 1). Thus, the equilibrium constants for these elimination reactions on the enzyme were probably not perturbed significantly from those in solution. The equilibrium constant for the reduction of enzyme-bound NAD+ by adenosine was 8, and the analogous constant for the reduction of the enzyme by S-adenosylhomocysteine was 4. The equilibrium constant for the reduction of NAD+ by a secondary alcohol in solution was 5 x 10(-5) at pH 7.0. Consequently, the reduction of enzyme-bound NAD+ by adenosine was 10(5)-fold more favorable than the reduction of free NAD+. The magnitude of the first-order rate constants for the interconversion of enzyme-bound intermediates varied over a relatively small range (3-80 s-1). Similarly, the magnitude of the equilibrium constants among enzyme-bound intermediates varied over a narrow range (0.3-10). These results were consistent with the overall reversibility of the reaction.
在pH 7.0和25℃条件下，通过停流荧光光谱法研究了S-腺苷同型半胱氨酸水解酶的动力学机制。利用该酶作用机制所提出的化学步骤（Palmer, J.L., and Abeles, R.H. (1979) J. Biol. Chem. 254, 1217 - 1226）确定了预稳态动力学步骤。S-腺苷同型半胱氨酸水解或合成的稳态动力学常数与根据预稳态速率常数计算得到的值高度一致。酶上3'-酮腺苷脱水生成3'-酮-4',5'-脱氢腺苷的平衡常数为3。酶上L-同型半胱氨酸加到S-3'-酮-4',5'-脱氢腺苷同型半胱氨酸的类似平衡常数为0.3。溶液中腺苷消除H2O的平衡常数为1.4（aH2O = 1）。因此，这些酶上消除反应的平衡常数可能与溶液中的平衡常数相比没有显著扰动。腺苷还原酶结合的NAD+的平衡常数为8，S-腺苷同型半胱氨酸还原酶的类似常数为4。在pH 7.0时，溶液中仲醇还原NAD+的平衡常数为5×10(-5)。因此，腺苷还原酶结合的NAD+比还原游离NAD+有利10(5)倍。酶结合中间体相互转化的一级速率常数大小在相对较小的范围内变化（3 - 80 s-1）。同样，酶结合中间体之间的平衡常数大小在狭窄范围内变化（0.3 - 10）。这些结果与反应的整体可逆性一致。
