Contributions of Active Site Residues to the Partial and Overall Catalytic Activities of Human S-Adenosylhomocysteine Hydrolase

Abstract

Residues glutamate 156 (E156), aspartate 190 (D190), asparagine 181 (N181), lysine 186 (K186), and asparagine 191 (N191) in the active site of S-adenosylhomocysteine (AdoHcy) hydrolase have been mutated to alanine (A). AdoHcy hydrolase achieves catalysis of AdoHcy hydrolysis to adenosine (Ado) and homocysteine (Hcy) by means of a redox partial reaction (3'-oxidation of AdoHcy at the beginning and 3'-reduction of Ado at the end of the catalytic cycle) spanning an elimination/addition partial reaction (elimination of Hcy from the oxidized substrate and addition of water to generate the oxidized product), with the enzyme in an open NAD(+) form in the ligand-free state and in a closed NADH form during the elimination/addition partial reaction. Mutation K186A reduces the rate of a model enzymatic reaction for the redox partial reaction by a factor of 280000 and the rate of a model reaction for the elimination/addition partial reaction by a factor of 24000, consistent with a primary catalytic role in both partial reactions as a proton donor/acceptor at the 3'-OH/3'-keto center. Secondary roles for N181 and N191 in localizing the flexible side chain of K186 in a catalytically effective position are supported by rate reduction factors for N181A of 2500 (redox) and 240 (elimination/addition) and for N191A of 730 (redox) and 340 (elimination/addition). A role of D190 in orienting the substrate for effective transition-state stabilization is consistent with rate reduction factors of 1300 (redox) and 30 (elimination/addition) for D190A. Residue E156 may act to maintain K186 in the desired protonation state: rate deduction factors are 1100 (redox) and 70 (elimination/addition). The mutational increases in free energy barriers for k(cat)/K(M) are described by a linear combination of the effects for the partial reactions with the coefficients equal to the fractional degree that each partial reaction determines the rate for k(cat)/K(M). A similar linear equation for k(cat) overestimates the barrier increase by a uniform 5 kJ/mol, probably reflecting reactant-state stabilization by the wild-type enzyme that is abolished by the mutations.

S-Adenosylhomocysteine Hydrolase from <i>Corynebacterium glutamicum:</i> Cloning, Overexpression, Purification, and Biochemical Characterization

“…2 C). These amino acid residues are very similar to those reported for Pf SAHase [Tanaka et al, 2004] and for human SAHase [Elrod et al, 2002;Yamada et al, 2005]. All other amino acid residues implied in the active site of Cg SAHase (Ile54, Thr57, Gln59, Leu393, His399, Met404, and Phe408) are almost identical to those reported for Pf SAHase ( fig.…”

The Rationale for Targeting the NAD/NADH Cofactor Binding Site of Parasitic S-Adenosyl-L-Homocysteine Hydrolase for the Design of Anti-Parasitic Drugs

“…Type I inhibitor (NepA) and Type II inhibitor (EDDFHA) structure and their inactivation mechanisms (adopted from Elrod, et al 2002 37 ).…”

A Tribute to Ronald T. Borchardt—Teacher, Mentor, Scientist, Colleague, Leader, Friend, and Family Man

“…[113][114][115][116][117][118][119][120][121][122][123] The mechanism by which designed adenosine analogs inactivate AdoHcy hydrolase was also elucidated and provided better understanding of the enzyme's active site and catalytic mechanism. 112,119,[124][125][126][127][128][129][130][131][132][133] These advances were made possible through long standing and productive collaborations with Ron's fellow KU faculty members Professors Richard Schowen and Krzysztof Kuczera, and Professor P. Lynne Howell at the University of Toronto.…”