Kinetic, Association, and Structural Properties of Dehydrogenases

Oklahoma State University, Stillwater OK

Investigators

Abstract

Howard Spivey MCB 0080258 Compelling experimental evidence has accumulated indicating that the previously considered soluble enzymes are often associated with other enzymes in the same cell compartment or associated with membrane or cytoskeletal components, i.e., they are not independently positioned in a soluble phase of a cellular compartment (e.g., cytoplasm, mitochondria, nucleus or other organelle). In some cases, these interactions have been shown to be critical to cell survival even in the absence of the enzyme activity. Also in many cases, enzyme concentrations in vivo exceed those of their substrates, and thus the majority of a substrate would be expected to be enzyme bound. Furthermore, evidence in vitro and in vivo indicates that some metabolites can be directly transferred from the enzyme producing it to the enzyme utilizing it - a process called substrate channeling. The potential physiological advantages of these enzyme associations and substrate channeling are numerous and significant. For example, they allow local enzyme reactions to proceed independently of the substrate concentrations elsewhere in the same cellular compartment. Thus, a better understanding of these process and implications (e.g., micro-compartmentation of metabolism) is essential for better understanding of cell function. These studies focus on the channeling of NADH between dehydrogenase enzymes. This research project will pursue the following objectives: 1) Obtain direct spectroscopic evidence of the NADH channeling complex and elucidate the kinetic mechanisms; 2) Correlate the NADH channeling properties with molecular structures of the enzymes using molecular modeling and site directed mutagenesis methods; and 3) Test the physiological significance of NADH channeling by expression of enzyme mutants in yeast that lack surface components essential for association and channeling, but have significant enzymatic activity.