Dr. Colman's laboratory is also interested in mechanisms of catalysis and regulation of dehydrogenases. As examples, she is focusing on two distinct isocitrate dehydrogenases of mammalian tissues. Colman's laboratory 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 is known and studies are in progress to determine its tertiary structure by X-ray crystallography. A major tool being 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.
Studies are in progress to express, purify and characterize mutant enzymes.
In addition, affinity labeling by reactive nucleotide analogues synthesized in this laboratory are being used to locate sub-regions of the coenzyme site. The modified or cleaved peptides are isolated and their amino acid sequences determined. These possibilities are being 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. In addition, site-directed mutagenesis is being used to prepare enzyme in which one subunit at a time contains a single amino acid substitution. Relationships between the NAD and NADP enzymes may be found in the active site despite differences in their physicochemical properties.