Enzyme Structure-Function Relationships

点击翻译按钮获取中文摘要

9728162 Remington The primary focus of this investigation will be to determine three dimensional structures of enzymes that catalyze the deprotonation of a simple carbon acid, acetylCoenzyme A, as a first step in the overall reaction. The enzymes are allosteric (regulated) Azotobacter citrate synthase and Escherichia coli malate synthase, which are unrelated in amino acid sequence but catalyze virtually identical reactions. A wide variety of acetylCoenzyme A substrate analogs, that vary in their ability to mimic proposed reaction intermediates, are available or are straightforward to synthesize. The structures of complexes of these analogs with the two enzymes will be determined by high resolution X-ray crystallography, and the results will be compared to those already obtained for the same compounds bound to nonallosteric chicken citrate synthase, which have been determined at up to 1.5A resolution. In addition, some active-site mutants of chicken citrate synthase will be created and their structures determined in order to ascertain the functional role of certain amino acids. A minor focus of this project, the structure of yeast serine carboxypeptidase in complex with an extremely tight binding, new class of cytosolic proteinase inhibitor (25 kDa Ic, Ki = 10-10 M) will be determined. The complex has been crystallized and the structure will be determined by standard molecular replacement methods using the known structure of serine carboxypeptidase. The significance of the major focus of the project is that it is not understood how an enzyme can deprotonate a carbon atom without using unusual cofactors. This reaction does not take place in solution under physiological conditions in the absence of a catalyst. By "freezing" two unrelated enzymes that catalyze this reaction as the first step and comparing their structures, new insight will be generated as to the exact nature of the intermediate(s), leading to a more complete understanding of this crucial reaction. Compounds that are rela ted to the different proposed intermediates, but are unable to complete subsequent steps of the reaction, are available to make this study straightforward. The second approach will be to compare the active site structures of regulated citrate synthases with those of unregulated citrate synthases. Structural changes at the active site caused by the external effector (NADH) should yield additional clues as to how the intermediate is generated and thus how the reaction is catalyzed. Finally, mutant citrate synthases have already been generated that are severely impaired in one or more aspects of catalysis, and their structures, which have yet to be determined, are expected to be very informative. The significance of the minor focus of this project is that the serine carboxypeptidase inhibitor Ic has multiple roles in the cell as defined by genetic analysis, but is of unknown structure. Completion of the three dimensional structure of the complex of serine carboxypeptidase with the inhibitor will therefore reveal a novel structure, expanding the database of protein folds in general, and will additionally lead to a deeper understanding of the catalytic mechanism of serine carboxypeptidases, and how they recognize their unique substrates.