Glutamates 78 and 122 in the Active Site of Saccharopine Dehydrogenase Contribute to Reactant Binding and Modulate the Basicity of the Acid-Base Catalysts

Babak Andi,Paul F Cook,Devi K Ekanayake,Ann H West,Kostyantyn D Bobyk

doi:10.1074/jbc.m110.119826

Abstract

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.

Highlights

On the basis of the pH dependence of the kinetic parameters [9], dissociation constants for the competitive inhibitors [1], and isotope effects [9], a chemical mechanism has been proposed for Saccharopine dehydrogenase (SDH) [1, 10]
There are a number of ionizable residues in the active site, and a multiple sequence alignment of the SDH from C. albicans, Pichia guilliermondii, S. cerevisiae, A. fumigatus, and C. neoformans indicated that all are conserved in all five organisms, consistent with their importance in the mechanism
The E78Q mutant enzyme was eluted from the NiNTA column with buffer containing 150 –180 mM imidazole at pH 8, whereas all others eluted at Ն180 mM imidazole

Summary

EXPERIMENTAL PROCEDURES

Materials—L-Saccharopine, L-lysine, ␣-Kg, ampicillin, chloramphenicol, phenylmethylsulfonyl fluoride, horse liver alcohol dehydrogenase, and bakers’ yeast aldehyde dehydrogenase were obtained from Sigma. ␤-NADH, ␤-NAD, Luria-Bertani (LB) broth, LB-agar, and imidazole were purchased form U. In the direction of lysine formation, initial rates were measured for E78Q and E122Q mutant enzymes, as a function of saccharopine concentration (0.5–10 Km) at different fixed levels of NAD (0.5–10 Km). Experiments were carried out for only the single mutant proteins, varying ␣-Kg with the other two substrates fixed at saturation (Ն10 Km) For both double mutant enzymes, lysine inhibited the reaction at low concentrations of ␣-Kg, and V/K␣-Kg was not determined. Multiple Solvent Deuterium/Substrate Deuterium Kinetic Isotope Effects—Multiple isotope effects were determined in the direction of saccharopine formation, for all mutant enzymes, by direct comparison of the initial rates in H2O and D2O as above, varying lysine at a fixed saturating concentration of NADD and ␣-Kg (Ն10 Km). Molecular Graphics—The active site figure of SDH was prepared using PyMOL version 0.99 [18]

RESULTS

C NC NC NC NC

DISCUSSION