The mechanism of action of the fragile histidine triad, Fhit: isolation of a covalent adenylyl enzyme and chemical rescue of H96G-Fhit.
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.
Huang
Kaisheng
K
Department of Biochemistry, University of Wisconsin-Madison, 1710 University Avenue, Madison, Wisconsin 53726, USA.
Arabshahi
Abolfazl
A
Wei
Yaoming
Y
Frey
Perry A
PA
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D033963:Adaptor Protein Complex 3 / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D033963:Adaptor Protein Complex 3; D000249:Adenosine Monophosphate; D008274:Magnesium; D009363:Neoplasm Proteins; D000099170:Bis(5'-adenosyl)triphosphatase