Student Research - Biochemistry and Molecular Biology
Sophonie Jean
Definig the Role of "Second Sphere" Residues in the Activity of Glyoxasomal Malate Dehydrogenase
Abstract: Defining the Role of "Second Sphere" Residues in the Activity of Glyoxasomal Malate Dehydrogenase Sophonie Jean, & Ellis Bell Laboratory for Structural Biology, Biophysics and Bioinformatics, University of Richmond, Richmond, Virginia 23173 Malate Dehydrogenase catalyses the interconversion of Malate and Oxaloacetate with the concommitant oxidation and reduction of the cofactor NAD(H). Although the 3 dimensional structure has been known for many years and a catalytic mechanism proposed involving a histidine-aspartate pair in the active site, little is known about the roles that residues further from the active site may play in either the hydride transfer or proton abstraction steps of catalysis. Analysis of the active site cavity of the enzyme, both visually and by QM-MM approaches, led to the design of a number of "second sphere" mutants involving various hydrophilic residues located 5-10A from the site of catalysis. Mutants were constructed using quikchange mutagenesis and the resultant proteins purified using NiNTA affinity chromatography making use of the histidine tag introduced in the pQE 60 vector used for expression. While H220 and D193 play well characterized roles in Proton Abstraction, a series of other residues, N124, T152, T186, A183, G180, V164, N165, S166 and H90 have been mutated and appear to play roles in the hydride transfer part of the overall reaction. The activity of various mutants has been characterized using initial rate kinetic studies and Km and Vmax values determined. Activation energies have been calculated from the temperature dependence of Vmax and the stabilities of the protein determined using both heat inactivation and guanidine hydrochloride denaturation. It is clear that a number of residues within this second sphere contribute to both protein stability and activity. This work is supported by NSF Grant MCB 0448905 to EB.
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