KINETIC-STUDIES OF THYMIDINE PHOSPHORYLASE FROM MOUSE-LIVER
KINETIC-STUDIES OF THYMIDINE PHOSPHORYLASE FROM MOUSE-LIVER
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DOI：
10.1021/bi00345a011
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
CHA, S
中科院分区：
文献类型：
--
作者：
ILTZSCH, MH;ELKOUNI, MH;CHA, S
小鼠肝脏胸苷磷酸化酶的起始速率和产物抑制研究表明，该酶的基本反应机理为酶-磷酸盐-胸腺嘧啶末端复合体的快速平衡随机双双机理。胸腺嘧啶既表现为底物抑制，又表现为非线性产物抑制，即斜率和截距曲线与1/[胸腺嘧啶]呈非线性关系，说明酶上存在一个以上的胸腺嘧啶结合位点，当胸腺嘧啶与其中一个结合时，该酶被抑制。此外，胸腺嘧啶核苷和磷酸盐在胸腺嘧啶浓度高于60µm时都表现出“协同效应”，这表明该酶可能具有多个相互作用的变构和/或催化部位。该酶催化的脱氧核糖转移酶反应依赖于磷酸盐，需要非化学计量比的磷酸盐，并可通过酶结合的2-脱氧核糖-1-磷酸中间体进行。这些发现符合快速平衡的随机bi-bi机制，表明该酶的脱氧核糖转移涉及间接转移机制。这些结果有力地表明，磷酸化和脱氧核糖转移是由胸苷磷酸化酶上的同一位点催化的。
Initial velocity and product inhibition studies of thymidine phosphorylase from mouse liver revealed that the basic reaction mechanism of this enzyme is a rapid equilibrium random bi-bi mechanism with an enzyme-phosphate-thymine dead-end complex. Thymine displayed both substrate inhibition and nonlinear product inhibition, i.e., slope and intercept replots vs. 1/[thymine] were nonlinear, indicating that there is more than one binding site on the enzyme for thymine and that when thymine is bound to one of these sites, the enzyme is inhibited. Furthermore, both thymidine and phosphate showed "cooperative effects" in the presence of thymine at concentrations above 60 .mu.M, suggesting that the enzyme may have multiple interacting allosteric and/or catalytic sites. The deoxyribosyl transferase reaction catalyzed by this enzyme is phosphate-dependent, requires nonstoichiometric amounts of phosphate, and can proceed by an "enzyme-bound" 2-deoxyribose 1-phosphate intermediate. These findings are in accord with the rapid equilibrium random bi-bi mechanism and demonstrate that deoxyribosyl transfer by this enzyme involves an indirect-transfer mechanism. These results strongly suggest that phosphoryolysis and deoxyribosyl transfer are catalyzed by the same site on thymidine phosphorylase.