STRUCTURE AND MECHANISMS OF FAMILY 18 CHITINASES

18 族几丁质酶的结构和机制

• 批准号: 2850574
• 负责人: JEFFRY D. MADURA
• 金额: $ 20.04万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2850574
• 关键词: N acetylglucosamine; O glycosidase; X ray crystallography; acidity /alkalinity; active sites; chemical binding; chemical kinetics; chitin; computer simulation; enzyme activity; enzyme mechanism; enzyme model; enzyme substrate complex; fluorescence spectrometry; hydrolysis; ionic bond; ionic strengths; molecular cloning; molecular dynamics; molecular polarity; polysaccharides; quantum chemistry; site directed mutagenesis; structural biology

This research will elucidate the biochemistry and structural biology for a class of enzymes and proteins known as glycosyl hydrolases. We will specifically study glycosyl hydrolases that hydrolyze chitin and chitin- like polymers into smaller saccharides and ultimately to monosaccharide. This is performed by a set of enzymes that cleave either in the middle of the polysaccharide or by releasing mono-or disaccharides from either the non-reducing or reducing end of the polysaccharide and its fragments. In order to accomplish our goal, we plan to use computational chemistry methods coupled with molecular and structural biological techniques to study Chitinase A, Brp39, ( breast regression protein ), and Chitobiase, all members of the same chitinolytic Family. For example molecular mechanics/dynamics will be used to investigate the binding of polysaccharides to Chitinase A while coupled quantum mechanics/molecular mechanics will be used to study the catalytic reaction mechanism. Molecular biology and biochemistry will be used to validate the computational results as well as produce Chitinase A mutants that bind a polysaccharide without cleaving. A three-dimensional crystal structure of this inactive Chitinase A mutant with the bound substrate will be determined by x-ray crystallography. Thus the combined computational, molecular biological and structural results of this effort will yield a clear detailed molecular picture and understanding of the various interactions responsible of the binding of a polysaccharide to Chitinase A and an unambiguous picture of the catalytic reaction mechanism. Similar procedures will be followed in the study of Brp39 and Chitobiase. The new knowledge from this work will be applicable across several of the over 50 glycosyl hydrolase Families. This is because there is 1] conservation of the vital catalytic and other active site residues; 2] common structural evolution of glycosidases with the (beta/alpha)8-barrel folding motif; and 3] similar acid/base or substrate-assisted catalytic reaction mechanisms. These results are medically relevant for understanding lysosomal storage disease mechanisms. For example, most of the lysosomal hydrolases whose genetic deficiencies cause devastating tissue storage diseases are glycosidases, including hexosaminidase defects being responsible for Tay-Sachs or Sandhoff disease. Additionally, Brp39 is in a subgroup of these proteins that no longer show catalytic activity but which work in a variety of developmental and differentiation processes, most likely via an ability to bind oligomers of chitin. Finally, these results are of biotechnological relevance such as in the development of antifungal agents.

这项研究将阐明生物化学和结构生物学 一类称为糖基水解酶的酶和蛋白质。 我们将 专门研究水解几丁质和几丁质的糖基水解酶 像聚合物一样成较小的糖，最终变成单糖。 这是由一组在中间切割的酶 多糖或通过从任何一个释放单糖或二糖 多糖的非还原或还原端 碎片。 为了实现我们的目标，我们计划使用 计算化学方法与分子和结构 研究几丁质酶A，BRP39的生物学技术（乳房回归 蛋白质）和Chitobiase，同一丁丁蛋白水解家族的所有成员。 例如，分子力学/动力学将用于研究 多糖与几丁质酶的结合量偶联量子 力学/分子力学将用于研究催化 反应机制。 分子生物学和生物化学将用于 验证计算结果并产生几丁质酶A 结合多糖而无需切割的突变体。三维 这种无活性几丁质酶的晶体结构是一个与结合的突变体 底物将通过X射线晶体学确定。 因此 合并的计算，分子生物学和结构结果 这项工作将产生明确的详细分子图片，并且 理解负责结合的各种相互作用 几糖酶A的多糖和明确的图片 催化反应机制。 类似的程序将遵循 BRP39和壳聚糖症的研究。 这项工作的新知识 将适用于50多个糖基水解酶中的几个 家庭。 这是因为有1]至关重要 催化和其他活性位点残留； 2]常见的结构进化 带有（β/α）8桶折叠基序的糖苷酶的糖苷酶；和3] 类似的酸/碱或底物辅助催化反应机制。 这些结果与理解溶酶体储存具有医学上的相关 疾病机制。 例如，大多数溶酶体水解酶 遗传缺陷引起毁灭性组织存储疾病是 糖苷酶，包括己糖胺酶缺陷。 Tay-Sachs或Sandhoff病。此外，brp39位于 这些不再显示催化活性但在 各种发展和差异化过程，很可能 通过结合几丁质的低聚物的能力。 最后，这些结果是 生物技术相关性，例如抗真菌的发展 代理商。