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

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• 作者：Philip Elrod ; Jinsong Zhang ; Xiaoda Yang ; Dan Yin ; Yongbo Hu ; Ronald T. Borchardt ; and Richard L. Schowen
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：June 25, 2002
• 年：2002
• 卷：41
• 期：25
• 页码：8134 - 8142
• 全文大小：135K
• 年卷期：v.41,no.25(June 25, 2002)
• ISSN：1520-4995

文摘

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) hydrolasehave 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 thebeginning and 3'-reduction of Ado at the end of the catalytic cycle) spanning an elimination/additionpartial reaction (elimination of Hcy from the oxidized substrate and addition of water to generate theoxidized product), with the enzyme in an open NAD⁺ form in the ligand-free state and in a closed NADHform during the elimination/addition partial reaction. Mutation K186A reduces the rate of a model enzymaticreaction for the redox partial reaction by a factor of 280000 and the rate of a model reaction for theelimination/addition partial reaction by a factor of 24000, consistent with a primary catalytic role in bothpartial reactions as a proton donor/acceptor at the 3'-OH/3'-keto center. Secondary roles for N181 andN191 in localizing the flexible side chain of K186 in a catalytically effective position are supported byrate 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 deductionfactors are 1100 (redox) and 70 (elimination/addition). The mutational increases in free energy barriersfor k_cat/K_M are described by a linear combination of the effects for the partial reactions with the coefficientsequal to the fractional degree that each partial reaction determines the rate for k_cat/K_M. A similar linearequation 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.