Studies of the kinetic mechanism of orotate phosphoribosyltransferase from yeast.
Studies of the kinetic mechanism of orotate phosphoribosyltransferase from yeast.
复制标题
酵母乳清酸磷酸核糖基转移酶动力学机制的研究。
DOI：
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发表时间：
1979
影响因子：
4.8
通讯作者：
D. Sloan
中科院分区：
文献类型：
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作者：
J. Victor;L. Greenberg;D. Sloan
以酵母为原料均相制备的螺旋状磷酸核糖基转移酶催化反应的动力学分析表明，该反应可能采用毕比乒乓动力学机制进行。这一解释是基于初始速率测量和在过量镁离子存在下的正向磷酸核糖基转移和反向焦磷分解反应的产物抑制研究。因此，生理上的磷酸核糖基转移反应可能是由磷酸核糖基1′焦磷酸（PRib-PP）与焦磷酸（PPi）从a-1′位置释放形成活化的核糖基磷酸酶中间体而引发的。反应的后半部分是j？orotidine Y-phosphate （OMP）与酶上的核糖磷酸部分之间的- 1 ' -糖苷键形成orotidine Y-phosphate。每个半反应都用标记的[' 4C]旋旋酸酯和[32P]PPi进行了表征，结果表明该酶首先催化了[' 4C]旋旋酸酯和OMP之间的标记交换，其次催化了[32P]PPi和P-Rib-PP之间的标记交换。因为立体化学反转是整个反应的结果，所以这种乒乓反应必然包含一个比双位移更复杂的机制。我们认为酶结合的核苷磷酸以碳离子的形式存在。测定了P-Rib-PP、orotate、PPi和OMP的Michaelis常数（K…）分别为38 + 6 PM、35 K 9 PM、96 f 6 PM和8 + 2 PM。利用氟旋酸酯得到的P-Rib-PP的初速度图和K值与用氟旋酸酯观察到的相似。氟旋光酸酯的K…值为27f5pm，与旋光酸酯的值相似。根据正反反应的动力学数据计算了所有反应物的生成物抑制常数Ki。这些Ki值被计算为42和5 pM， 63 f 2 pM， 131 + 50 pM和10 f 2 pM的P-Rib-PP，旋转，f ！分别取Pi和OMP，并与Km和V一起，计算出可逆磷酸核糖基转移反应的平衡常数为0.49。
Kinetic analysis of the reaction catalyzed by a homogeneous preparation of orotate phosphoribosyltransferase from yeast has revealed that the reaction may proceed through the use of a Bi Bi Ping Pong kinetic mechanism. This interpretation is based on initial rate measurements and product inhibition studies of both the forward phosphoribosyl transfer and the reverse pyrophosphorolysis reactions in the presence of excess magnesium ion. The physiological phosphoribosyl transfer reaction may thus be initiated by the formation of an activated ribosylphosphate-enzyme intermediate from phosphoribosyl l’-pyrophosphate (PRib-PP) with the release of pyrophosphate (PPi) from the a-1’ position. The second half of the reaction involves the formation of a j?-l’-glycosidic bond between orotate and the ribose phosphate moiety on the enzyme to form orotidine Y-phosphate (OMP). Each half-reaction was characterized using labeled [‘4C]orotate and [32P]PPi and the enzyme was shown to catalyze an exchange of label, first, between [‘4C]orotate and OMP in the absence of P-Rib-PP, and second, between [32P]PPi an d P-Rib-PP in the absence of orotate. Because stereochemical inversion results from the overall reaction, this ping-pong reaction must involve a mechanism more complicated than a double displacement. We propose that the enzyme-bound ribosylphosphate exists as a carbonium ion. Michaelis constants (K,,,) for P-Rib-PP, orotate, PPi, and OMP were determined to be 38 + 6 PM, 35 k 9 PM, 96 f 6 PM, and 8 + 2 pM, respectively. The utilization of Hluoro-orotate produced initial velocity plots and a K,,, value of P-Rib-PP analogous to those observed with orotate. A K,,, value of 27 f 5 pM calculated for fluoroorotate was similar to the value determined for orotate. Product inhibition constants (Ki) for all of the reactants were calculated from the kinetic data for both the forward and reverse reactions. These values of Ki were calculated to be 42 & 5 pM, 63 f 2 pM, 131 + 50 pM, and 10 f 2 PM for P-Rib-PP, orotate, F!Pi, and OMP, respectively, and were used along with the Km and V,, values to calculate a value of 0.49 for the equilibrium constant for the reversible phosphoribosyl transfer reaction.