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

Abstract

The human fragile histidine triad protein Fhit catalyzes the Mg(2+)-dependent hydrolysis of P(1)-5'-O-adenosine-P(3)-5'-O-adenosine triphosphate, Ap(3)A, to AMP and ADP. The reaction is thought to follow a two-step mechanism, in which the complex of Ap(3)A and Mg(2+) 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'-(3)H]-Ap(3)A 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 MgAp(3)A. 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 MgAp(3)A. The results indicate that Mg(2+) in the reaction of Ap(3)A 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.

Loss of Heterozygosity Patterns Provide Fingerprints for Genetic Heterogeneity in Multistep Cancer Progression of Tobacco Smoke–Induced Non–Small Cell Lung Cancer

“…The prominent putative Table 3. Tobacco smoke-exposed BECs, adenocarcinoma, and SCC exhibit common and distinct allelic loss patterns (30,31). Frequent allelic losses, homozygous deletions, and aberrant transcripts of the FHIT gene have been described in a high percentage of lung tumors (30).…”

Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

“…Assay of Ap 3 A and Ap 4 A Hydrolysis-A coupled assay was applied to determine the rate of hydrolysis of Ap 3 A and Ap 4 A as described for human Fhit (18). Initial rates were measured in a Cary 50 spectrophotometer at 25°C.…”

“…Like Hint1, Fhit is also a homodimer and tumor suppressor (17). Human Fhit is a Ap 3 A hydrolyase as well as phosphoramidase (18,19). The precise mechanism of action by which it affects tumor development is not well understood, although site-directed mutagenesis studies have indicated that the tumor suppressor function of Fhit is likely not dependent on its Ap 3 A hydrolase activity (20).…”

Immunohistochemical characterization of FHIT expression in normal human tissues

“…At the cellular level, FHIT has been shown to induce apoptosis and retard tumor cell proliferation in vitro and in vivo [25,26]. Moreover, it has been established that the FHIT gene encodes a diadenosine polyphosphate (Ap3A) hydrolase [16,19,27]. Therefore, FHIT product is believed to have hydrolytic activity.…”

“…The reaction is thought to follow a two-step mechanism, in which a complex of Ap3A and Mg 2+ reacts in the fi rst 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 [27]. Another suggestion hypothesized by Zhao et al was that the executive mode of the nuclear localization of FHIT protein is due to the binding with other eff ector molecules that have been reported, such as tubulin, Ubc9, Nit, Hint, CDK7, and so on [24].…”