Mammalian tissues contain both an NADP-specific and an NAD-dependent isocitrate dehydrogenase which exhibit similarities in their catalytic reactions, but differ in their physical characteristics as well as in their mode of regulation. The pig heart NADP-dependent enzyme is a dimer of identical subunits and is not subject to control by modifiers; whereas the NAD-specific enzyme from the same species and tissue is activated by ADP and is composed of 3 types of subunits present in the ratio of 2alpha:1beta:1gamma. This study aims at identifying and ascertaining the role of those amino acids critical for function in both enzymes. The primary structure of the pig heart NADP enzyme has recently been completed in our collaborative studies involving nucleotide and protein sequencing; and studies are in progress to determine its tertiary structure by X-ray crystallography. For this NADP-specific enzyme in solution, we now aim to locate those regions in which metal-isocitrate and coenzyme bind and to evaluate the residues which contribute directly to binding and/or catalysis. The major tool to be used is site-directed mutagenesis, with the target sites for mutation selected by analysis of affinity modification results, crystal structures and sequence alignments with functionally comparable enzymes. Mutant enzymes will be expressed, purified and characterized. In addition, affinity labeling by reactive nucleotide analogues synthesized in this laboratory will be used to locate sub-regions of the coenzyme site; and affinity cleavage by metal- isocitrate will help to localize the substrate binding site. The modified or cleaved peptides will be isolated and their amino acid sequences determined. For the NAD enzyme, which has 2 binding sites/enzyme tetramer for all ligands, a major focus will be on assessing the roles of the dissimilar subunits. Substantial segments of these subunits have already been sequenced and this work will continue. Subunit types of the NAD-enzyme may have specialized functions; alternatively, all subunits may have the potential to bind every type of ligand but only half may actually bind at a time. These possibilities will be evaluated by identification of the subunit types affected by site-specific labels and affinity cleavage, and of the sequences of modified peptides derived from the distinct subunits. Kinetic and ligand binding studies on modified enzymes will also be conducted. NMR studies will use 113Cd and 13C-enriched substrates to explore the metal-substrate sites of the NAD enzyme for comparison with the NADP-enzyme. Relationships between the NAD and NADP enzymes may be found in the active site despite differences in their physicochemical properties.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK039075-07
Application #
2140769
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1987-07-01
Project End
1996-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
7
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Delaware
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
059007500
City
Newark
State
DE
Country
United States
Zip Code
19716
Soundar, S; Danek, B L; Colman, R F (2000) Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase. J Biol Chem 275:5606-12
Grodsky, N B; Soundar, S; Colman, R F (2000) Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase. Biochemistry 39:2193-200
Huang, Y C; Soundar, S; Colman, R F (2000) Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenase. Protein Sci 9:104-11
Chen, H; Huang, Y C; Colman, R F (1998) Identification of the subunit and important target peptides of pig heart NAD-dependent isocitrate dehydrogenase modified by the affinity label adenosine 5'-O-[S-(4-bromo-2, 3-dioxobutyl)thiophosphate]. Biochemistry 37:6541-51
Colman, R F (1997) Chemical arrows for enzymatic targets. FASEB J 11:217-26
Huang, Y C; Kumar, A; Colman, R F (1997) Identification of the subunits and target peptides of pig heart NAD-specific isocitrate dehydrogenase modified by the affinity label 8-(4-bromo-2,3-dioxobutylthio)NAD. Arch Biochem Biophys 348:207-18
Soundar, S; Jennings, G T; McAlister-Henn, L et al. (1996) Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli. Protein Expr Purif 8:305-12
Ehrlich, R S; Colman, R F (1995) Cadmium-113 and magnesium-25 NMR study of the divalent metal binding sites of isocitrate dehydrogenases from pig heart. Biochim Biophys Acta 1246:135-41
Huang, Y C; Haselbeck, R J; McAlister-Henn, L et al. (1995) N-ethylmaleimide profiling of yeast NADP-dependent isocitrate dehydrogenase. Arch Biochem Biophys 316:485-92
Kumar, A; Colman, R F (1994) 8-(4-Bromo-2,3-dioxobutylthio)NAD: a new affinity label for NAD-specific isocitrate dehydrogenase. Arch Biochem Biophys 308:357-66

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