Mammalian tissues contain an NADP- and an NAD-specific isocitrate dehydrogenase which both catalyze a metal-dependent oxidative decarboxylation of isocitrate, but differ in their affinities for substrate, their mode of regulation and their structures. 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 and in elucidating the structural basis for kinetic differences between the two enzymes. The sequence of the pig heart NADP enzyme is now known; and good progress has been made in determining its tertiary structure by X-ray crystallography. For this NADP-specific enzyme in solution, we aim to identify amino acids contributing to catalysis and/or binding of metal-isocitrate and coenzyme. 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 in E. coli, purified and characterized. Affinity labeling by reactive nucleotide analogues synthesized in this laboratory will also be used to locate sub-regions of the coenzyme site. For the NAD enzyme, we will focus on the roles of the dissimilar subunits, which have recently been sequenced. The enzyme has 2 binding sites/enzyme tetramer. 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 (and peptides) affected by site-specific labels and affinity cleavage, and by studying the reassembly of enzyme from unmodified or affinity labeled separated subunits. Striking similarities exist between the NADP-dependent isocitrate dehydrogenase of pig and human hearts, suggesting that these studies will be relevant for understanding human cardiac energy metabolism.
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