Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani
Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani
复制标题
DOI：
10.1021/bi0158730
复制
发表时间：
2002-03-26
期刊：
影响因子：
2.9
通讯作者：
Ullman, B
中科院分区：
文献类型：
--
作者：
Bashor, C;Denu, JM;Ullman, B
腺嘌呤磷酸核糖基转移酶（APRT，EC 2.4.2.7）催化腺嘌呤从α-D-5-磷酸核糖基-1-焦磷酸（PRPP）的可逆磷酸核糖基化以形成AMP和PPj。来自杜氏利什曼原虫的二聚APRT酶（LdAPRT）的三维结构与I型磷酸核糖基转移酶家族的其它成员具有许多相似性，但没有揭示催化的结构基础（菲利普斯，C. L.，Ullman，B.，布伦南河，巴西-地G.，和Hill，C. P.（1999）EMBO J. 18，3533-3545）。为了解决这个问题，进行酶的稳态和瞬时动力学分析以确定催化机理。研究表明，LdAPRT遵循有序的顺序机制，其中PRPP是第一个结合的底物，NIP是最后一个离开的产物。平衡同位素交换和荧光结合的研究，提供了解离常数的LdAPRT-PRPP和LdAPRT-AMP二元复合物证实了这种机制模型。前稳态动力学分析的正向反应揭示了一个突发的产品形成表明磷酸核糖转移进展迅速，相对于一些限速产品释放事件。瞬时荧光竞争实验能够测量二元复合物解离的速率，该速率涉及AMP释放作为正向反应的速率限制。产物三元复合物形成的动力学进行了评价，使用荧光团福霉素AMP和建立的速率常数焦磷酸盐结合的LdAPRT-福霉素AMP复合物。两者合计，这些数据使一个有序的双双动力学机制LdAPRT中的所有速率常数进行测量或计算的完整配方。
Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PPj. Three-dimensional structures of the dimeric APRT enzyme from Leishmania donovani (LdAPRT) bear many similarities to other members of the type I phosphoribosyltransferase family but do not reveal the structural basis for catalysis (Phillips, C. L., Ullman, B., Brennan, R. G., and Hill, C. P. (1999) EMBO J. 18, 3533-3545). To address this issue., a steady state and transient kinetic analysis of the enzyme was performed in order to determine the catalytic mechanism. Initial velocity and product inhibition Studies indicated that LdAPRT follows an ordered sequential mechanism in which PRPP is the first substrate to bind and NIP is the last product to leave. This mechanistic model was substantiated by equilibrium isotope exchange and fluorescence binding studies, which provided dissociation constants for the LdAPRT-PRPP and LdAPRT-AMP binary complexes. Pre-steady-state kinetic analysis of the forward reaction revealed a burst in product formation indicating that phosphoribosyl transfer proceeds rapidly relative to some rate-limiting product release event. Transient fluorescence competition experiments enabled measurement of rates of binary complex dissociation that implicated AMP release as rate-limiting for the forward reaction. Kinetics of product ternary complex formation were evaluated using the fluorophore formycin AMP and established rate constants for pyrophosphate binding to the LdAPRT-formycin AMP complex. Taken together, these data enabled the complete formulation of an ordered bi-bi kinetic mechanism for LdAPRT in which all of the rate constants were either measured or calculated.