Contributions of active site residues to the partial and overall catalytic activities of human S-adenosylhomocysteine hydrolase.
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.
Elrod
Philip
P
Departments of Molecular Biosciences and Pharmaceutical Chemistry, Simons Research Laboratories, The University of Kansas, Lawrence, KS 66047, USA.
Zhang
Jinsong
J
Yang
Xiaoda
X
Yin
Dan
D
Hu
Yongbo
Y
Borchardt
Ronald T
RT
Schowen
Richard L
RL
D043383:Adenosylhomocysteinase; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017354:Point Mutation; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; D006867:Hydrolases; D043383:Adenosylhomocysteinase