The Mechanism of Action of the Fragile Histidine Triad, Fhit: Isolation of a Covalent Adenylyl Enzyme and Chemical Rescue of H96G-Fhit

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• 作者：Kaisheng Huang ; Abolfazl Arabshahi ; Yaoming Wei ; and Perry A. Frey
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：June 15, 2004
• 年：2004
• 卷：43
• 期：23
• 页码：7637 - 7642
• 全文大小：66K
• 年卷期：v.43,no.23(June 15, 2004)
• ISSN：1520-4995

文摘

The human fragile histidine triad protein Fhit catalyzes the Mg²⁺-dependent hydrolysis ofP¹-5'-O-adenosine-P³-5'-O-adenosine triphosphate, Ap₃A, to AMP and ADP. The reaction is thought tofollow a two-step mechanism, in which the complex of Ap₃A and Mg²⁺ reacts in the first step with His96of the enzyme to form a covalent Fhit-AMP intermediate and release MgADP. In the second step, theintermediate 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 analogousmechanism, 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). Directevidence in support of this mechanism is reported herein. Reaction of Fhit with [8,8'-³H]-Ap₃A anddenaturation of the enzyme in the steady state leads to protein-bound tritium corresponding to 11% of theactive sites. Similar experiments with the poor substrate MgATP leads to 0.9% labeling. The mutatedprotein H96G-Fhit is completely inactive against MgAp₃A. However, it is chemically rescued by freehistidine. H96G-Fhit also catalyzes the hydrolysis of adenosine-5'-phosphoimidazolide, AMP-Im, and ofadenosine-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 asefficiently as the hydrolysis of MgAp₃A. The results indicate that Mg²⁺ in the reaction of Ap₃A is requiredfor the first step, the formation of the covalent intermediate Fhit-AMP, and not for the hydrolysis of theintermediate in the second step.