This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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