ACTIVE SITE OF NAD(H) DEPENDENT ENZYMES

NAD(H)是多种酶的辅酶。

NAD(H) functions as the coenzyme of a large number of enzymes. In addition to its well documented roles in oxidation-reduction reactions, NAD(H) is also involved in many aspects of metabolic regulation which have only recently been recognized. An understanding of the structure of these enzymes will be important for the understanding of those biochemical processes associated with these enzymes. However, among all NAD(H) dependent enzymes, only the structure of a few simple oxido-reduction enzymes have been investigated. In this application we propose to investigate the active site of two NAD(H) dependent enzymes, bovine heart mitochondrial NADH dehydrogenase, and NADH-NAD+ transhydrogenase by affinity labeling techniques. Both of these enzymes are important enzymes involved in the cellular energy production process. The mitochondrial NAD(H) dehydrogenase has also been shown to take part in mechanisms inducing cardiac oxygen toxicity and 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity related to Parkinson Disease. Although many researchers have shown great interest in these enzymes, no structural information is yet available for either enzyme because of the heterogenity of these enzyme preparations. In this proposed project, the active sites of these enzymes will be identified by labeling with radioactive derivatives of six affinity probes of NAD+. The peptides modified by these probes will be characterized by microsequencing, amino acid sequences of analysis, and fast atom bombardment mass spectrometry. The amino acid sequences of the modified peptides of three enzymes will be compared to those at the active site regions of well characterized NAD(H)- dependent enzymes. This investigation will identify important regions of these two enzymes. The information generated through this study will be valuable for further understanding the molecular mechanism of the reactions these enzymes catalyze. Furthermore, this structural information will be very useful for future structure-function study of these two enzymes by molecular cloning and site specific mutagenesis experiments.