Mammalian tissues contain both a TPN-specific and a DPN- dependent isocitrate dehydrogenase which exhibit similarities in their catalytic reactions, but differ markedly in their physical characteristics as well as in their mode of regulation. The pig heart TPN-dependent enzyme is a dimer of identical subunits and is not subject to control by modifiers; whereas the DPN-specific enzyme from the same species and tissue is markedly affected by ADP and ATP, and is composed of 3 distinct types of subunits present in the ratio of 2 alpha:1 beta:1 gamma. This study aims at identifying and ascertaining the role of those amino acids critical for function in the non-regulatory and allosteric enzymes. For both enzymes the residues essential for catalysis and regulation will be elucidated by affinity labeling using substrate analogues such as 3-bromo-2-ketoglutarate and new reactive nucleotide analogues synthesized in this laboratory, as well as by limited chemical modification with group specific reagents. The modified peptides will be isolated and their amino acid sequences determined. The kinetic and ligand binding characteristics of the altered enzymes will be measured in order to evaluate the normal role of the modified amino acid residues. Native DPN-enzyme has 2 binding sites/enzyme tetramer for all ligands tested. Subunit types of the DPN-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 alternate possibilities will be evaluated by affinity labeling of specific sites coupled with an identification of the subunits. Hybrid enzymes, composed of chemically modified and native subunits, will be prepared and their catalytic and binding properties will be compared with those of native and completely modified enzymes. Crosslinking experiments will be conducted to elucidate the arrangement of subunit types within the native DPN enzyme. NMR studies will examine the conformations of nucleotides bound to the DPN-enzyme by transferred nuclear Overhauser effects for comparison with those we have previously detected for the TPN-enzyme. Relationships between the DPN- and TPN-enzymes may exist in the region of the active site despite differences in their physicochemical properties. Striking similarities exist between the TPN-dependent isocitrate dehydrogenase of pig and human hearts, suggesting that many of the results obtained from the proposed studies will be relevant for the understanding of human cardiac energy metabolism.
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