pH-dependence in the hydrolytic action of the human fragile histidine triad

Abstract

The human fragile histidine triad protein (Fhit) is a member of the HIT family of enzymes, which catalyze hydrolysis or nucleotidyltransfer reactions of dinucleoside polyphoshates. Fhit catalyzes the magnesium ion-dependent hydrolysis of P1-5′-O-adenosine-P3-5′-O- adenosine triphosphate (Ap3A) to adenosine-5′-O-phosphate (AMP) and the magnesium complex of adenosine-5′-O-diphosphate (ADP) by a double displacement mechanism, with the formation of an adenylyl enzyme as an intermediate. Fhit also catalyzes the hydrolysis of adenosine-5′- phosphoimidazolide (AMP-Im) and adenosine-5′-phospho-N-methylimidazolide (AMP-N-MeIm). The pH-dependence of these reactions is reported herein, in which the principal conclusions are as follows: The action of wild-type Fhit on MgAp3A is diffusion-limited and on AMP-Im and AMP-N-MeIm largely diffusion-limited and largely pH-independent. The actions of specifically mutated H96G-Fhit on AM-Im, and on AMP-N-MeIm, are not diffusion-limited and are pH-dependent. The actions of mutated forms of Fhit, H94G-Fhit, H98G-Fhit, and H94/98G-Fhit, are also not diffusion-limited and are pH-dependent. Log plots of kinetic parameters against pH show breaks that indicate a group on the enzyme must be protonated for maximal activity. Extensive analysis shows that the imidazole ring of His94 is not essential for the hydrolysis of MgAp3A or AMP-imidizolides, and the imidazole ring of His98 engages in binding the substrates. In the hydrolysis of AMP-Im, Fhit and its His94, His96, and His98 variants bind the monoanionic form of AMP-Im, and the proton required for formation of imidazole in the hydrolytic process originates with an acid/base group of the enzyme. Fhit and several variants also catalyze the hydrolysis of p-nitrophenyl-AMP. © Wiley-VCH Verlag GmbH & Co. KGaA, 2005.