pH-Dependence in the Hydrolytic Action of the Human Fragile Histidine Triad

✍ Scribed by Kaisheng Huang; Abolfazl Arabshahi; Perry A. Frey

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 152 KB
• Volume: 2005
• Category: Article
• ISSN: 1434-193X
• DOI: 10.1002/ejoc.200500499

✦ Synopsis

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 P^1–^5′‐O‐adenosine‐P^3–^5′‐O‐adenosine triphosphate (Ap~3~A) 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 MgAp~3~A 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 MgAp~3~A 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, 69451 Weinheim, Germany, 2005)