Kinetic, Association, and Structural Properties of Dehydrogenases

Howard SpiveyMCB 0080258令人信服的实验证据表明，以前认为的可溶酶往往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在细胞室(例如细胞质、线粒体、细胞核或其他细胞器)的可溶相中。在某些情况下，即使在没有酶活性的情况下，这些相互作用也被证明是细胞生存的关键。此外，在许多情况下，体内的酶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。这些酶联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隔间中其他地方的底物浓度而进行。因此，更好地了解这些过程和含义(例如，代谢的微区隔)对于更好地了解细胞功能是至关重要的。这些研究主要集中在脱氢酶之间的NADH通道。本研究项目将追求以下目标：1)获得NADH通道复合体的直接光谱证据并阐明其动力学机制；2)利用分子模拟和定点突变方法将NADH通道特性与酶的分子结构相关联；以及3)通过在酵母中表达缺乏结合和通道所必需的表面成分但具有显著酶活性的酶突变体来测试NADH通道的生理意义。

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.