Glutamates 78 and 122 in the active site of saccharopine dehydrogenase contribute to reactant binding and modulate the basicity of the acid-base catalysts.
Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give l-lysine and alpha-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, Glu(78) and Glu(122), 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 pK(a) 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 alpha-ketoglutarate to E and E.NADH. Large changes in V/K(Lys), but not V, suggest that Glu(78) and Glu(122) contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pK(a) values observed in the V/K(Lys) pH-rate profile of the mutant enzymes suggests that the presence of Glu(78) and Glu(122) modulates the basicity of the catalytic groups.
Ekanayake
Devi K
DK
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
Andi
Babak
B
Bobyk
Kostyantyn D
KD
West
Ann H
AH
Cook
Paul F
PF
D000136:Acid-Base Equilibrium; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D018698:Glutamic Acid / Q000032:analysis*; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000096:biosynthesis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012443:Saccharopine Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D005973:Glutamine; D009243:NAD; D018698:Glutamic Acid; D012443:Saccharopine Dehydrogenases; D008239:Lysine; D000409:Alanine