64-15-3Relevant academic research and scientific papers
Glutamates 78 and 122 in the Active Site of Saccharopine Dehydrogenase Contribute to Reactant Binding and Modulate the Basicity of the Acid-Base Catalysts
Ekanayake, Devi K.,Andi, Babak,Bobyk, Kostyantyn D.,West, Ann H.,Cook, Paul F.
experimental part, p. 20756 - 20768 (2011/04/18)
Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give L-lysine and α-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu78 and Glu122, on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pKa values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of α-ketoglutarate to E and E·NADH. Large changes in V/K Lys, but not V, suggest that Glu78 and Glu122 contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pKa values observed in the V/KLys pH-rate profile of the mutant enzymes suggests that the presence of Glu78 and Glu122 modulates the basicity of the catalytic groups.
Thermodynamics of reactions catalysed by branched-chain-amino-acid transaminase
Tewari, Yadu B.,Goldberg, Robert N.,Rozzell, J. David
, p. 1381 - 1398 (2007/10/03)
Apparent equilibrium constants and calorimetric enthalpies of reaction have been measured for reactions catalysed by branched-chain-amino-acid transaminase. The following biochemical reactions have been studied at the temperature 298.15 K and in the pH ra
