ENZYMATIC REACTION MECHANISM FOR LYSINE 2, 3-AMINOMUTASE AND THE OTHER ENZYMES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 人脆性组氨酸三联体蛋白Fhit催化Mg 2+依赖的P1-5？O-腺苷-P3-5 O-腺苷三磷酸，Ap 3A，AMP和ADP。 该反应被认为是遵循一个两步机制，其中的Ap 3A和Mg 2+的复合物反应在第一步与His 96的酶形成共价Fhit？AMP中间体并释放MgADP。 在第二步中，中间FHIT？AMP水解为AMP和Fhit。 该机制受到Fhit与半乳糖-1-磷酸尿苷酰转移酶的链折叠相似性的启发，其通过类似的机制起作用，并且观察到在Fhit的作用中在磷处构型的总体保留（Abend，A.，加里森，P.N.，巴恩斯有限公司Frey，P.A.（1999）Biochemistry 38，3668-3676）。本文报告了支持这一机制的直接证据。Fhit与[8.8- 3 H] Ap 3A的反应和稳态下酶的变性导致对应于11%活性位点的蛋白质结合的氚。 用不良底物MgATP的类似实验导致0.9%标记。突变蛋白H96 G-Fhit对MgAp 3A完全无活性。 然而，它被游离组氨酸化学拯救。 H96 G-Fhit还催化腺苷-5？磷酸咪唑，AMP-Im和腺苷-5-磷酸-N-甲基咪唑，AMP-N-MeIm。 水解的AMP-Im和AMP-N-MeIm的H96 G-Fhit被认为是代表化学救援的共价Fhit？AMP中间体。 还发现野生型Fhit催化AMP-Im和AMP-N-MeIm的水解几乎与MgAp 3A的水解一样有效。结果表明，Mg 2+在Ap 3A反应的第一步，形成共价中间体Fhit？AMP，而不是用于第二步中中间体的水解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The human fragile histidine triad protein Fhit catalyzes the Mg2+-dependent hydrolysis of P1-5?-O-adenosine-P3-5?-O-adenosine triphosphate, Ap3A, to AMP and ADP. The reaction is thought to follow a two-step mechanism, in which the complex of Ap3A and Mg2+ reacts in the first step with His96 of the enzyme to form a covalent Fhit?AMP intermediate and release MgADP. In the second step the intermediate Fhit?AMP undergoes hydrolysis to AMP and Fhit. The mechanism is inspired by the chain-fold similarities of Fhit to galactose-1-phosphate uridylyltransferase, which functions by an analogous mechanism, and the observation of overall retention in configuration at phosphorus in the action of Fhit (Abend, A., Garrison, P.N., Barnes, L.D. and Frey, P.A. (1999) Biochemistry 38, 3668-3676). Direct evidence in support of this mechanism is reported herein. Reaction of Fhit with [8.8-3H]Ap3A and denaturation of the enzyme in the steady state, leads to protein bound tritium corresponding to 11% of the active sites. Similar experiments with the poor substrate MgATP leads to 0.9% labeling. The mutated protein H96G-Fhit is completely inactive against MgAp3A. However, it is chemically rescued by free histidine. H96G-Fhit also catalyzes the hydrolysis of adenosine-5?-phosphoimidazolide, AMP-Im, and of adenosine-5-phospho-N-methylimidazolide, AMP-N-MeIm. The hydrolyses of AMP-Im and of AMP-N-MeIm by H96G-Fhit are thought to represent chemical rescue of the covalent Fhit?AMP intermediate. Wild type Fhit is also found to catalyze the hydrolyses of AMP-Im and of AMP-N-MeIm nearly as efficiently as the hydrolysis of MgAp3A. The results indicate that Mg2+ in the reaction of Ap3A is required for the first step, the formation of the covalent intermediate Fhit?AMP and not for the hydrolysis of the intermediate in the second step.