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Defining the Role of “Second Sphere” Residues in the Activity of Glyoxasomal Malate Dehydrogenase
Author(s) -
Jean Sophonie,
Bell Ellis
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a643-c
Subject(s) - histidine , active site , malate dehydrogenase , chemistry , mutant , substrate (aquarium) , stereochemistry , citrate synthase , enzyme , biochemistry , dehydrogenase , mutagenesis , binding site , biology , ecology , gene
Malate Dehydrogenase catalyses the interconversion of Malate and Oxaloacetate using NAD(H). Although the 3‐D structure has long been known 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 hydride transfer or proton abstraction. Analysis of the active site cavity, both visually and by QM‐MM approaches, led to the construction of “second sphere” mutants involving residues located 5–10A from the site of catalysis using quickchange mutagenesis, and the resultant proteins purified using Ni‐NTA affinity chromatography making use of the histidine tag introduced in the pQE 60 vector. While H220 and D193 play well characterized roles in proton abstraction, other residues, N124, T152, T186, A183, G180, V164, N165, S166 and H90 have been mutated and appear to play various roles in the overall reaction. Several mutants appear to be affecting the pH dependence and substrate inhibition. For example, while the N165D mutant showed similar Vmax values at pH 7 & 8 there is a marked increase in the effects of substrate inhibition at pH 7 and a reversal of the pH effects on Km for oxaloacetate relative to the native protein or the S166A mutant. The N165D and the S166A mutants have been selected for crystal screening. It is clear that a number of residues within this second sphere contribute to protein stability and aid in the fine tuning of the regulatory effects of substrate inhibition and pH dependence. This work is supported by NSF Grant MCB 0448905 to EB.