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.
Showing the most recent 10 out of 26 publications
