Kinetic, Association, and Structural Properties of Dehydrogenases

令人信服的实验证据表明，先前认为的可溶性酶通常与同一细胞室中的其他酶相关，或与膜或细胞骨架成分相关，即，它们不是独立地定位于细胞室的可溶性相（例如，细胞质，线粒体，细胞核或其他细胞器）。在某些情况下，即使在缺乏酶活性的情况下，这些相互作用也被证明对细胞存活至关重要。此外，在许多情况下，体内酶的浓度超过其底物的浓度，因此大多数底物将被酶结合。此外，体外和体内的证据表明，一些代谢物可以直接从产生它的酶转移到利用它的酶，这一过程被称为底物通道。这些酶结合和底物通道的潜在生理优势是众多和显著的。例如，它们允许局部酶反应独立于同一细胞室中其他地方的底物浓度进行。因此，更好地理解这些过程和影响（例如，代谢的微区隔）对于更好地理解细胞功能是必不可少的。这些研究集中在脱氢酶之间NADH的通道。本研究项目将追求以下目标：1)获得NADH通道复合物的直接光谱证据并阐明其动力学机制；2)利用分子模型和位点定向诱变方法将NADH通道特性与酶的分子结构联系起来；3)通过在酵母中表达酶突变体来检测NADH通道的生理意义，这些突变体缺乏连接和通道必需的表面组分，但具有显著的酶活性。

Howard SpiveyMCB 0080258Compelling 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.