STRUCTURE AND MECHANISMS OF FAMILY 18 CHITINASES

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 具体研究水解甲壳质和甲壳质糖基水解酶， 比如聚合物分解成更小的单糖，最后变成单糖。 这是由一组酶来完成的，这些酶在中间切割， 或通过从多糖中释放单糖或二糖 多糖的非还原或还原端及其 片段 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 生物技术研究几丁质酶A，Brp 39，（乳房退化 蛋白质）和几丁质双酶，它们都是同一几丁质分解家族的成员。 例如，分子力学/动力学将用于研究 多糖与几丁质酶A的结合，同时偶联量子 力学/分子力学将用于研究催化 反应机理 分子生物学和生物化学将用于 验证了计算结果，并生产了几丁质酶A 结合多糖而不裂解的突变体。三维 这种结合的无活性几丁质酶A突变体的晶体结构 衬底将通过X射线晶体学确定。 因此 结合计算，分子生物学和结构的结果， 这项工作将产生一个清晰详细的分子图像， 理解负责结合的各种相互作用， 一个多糖几丁质酶A和一个明确的图片， 催化反应机理 类似的程序将在 Brp 39和壳聚糖酶的研究。 这项工作的新知识 将适用于超过50种糖基水解酶中的几种 家庭 这是因为有1]保护的重要 催化和其他活性位点残基; 2]共同结构进化 具有（β/α）8-桶折叠基序的糖苷酶;和3] 类似酸/碱或底物辅助催化反应机理。 这些结果对于理解溶酶体贮积具有医学意义 疾病机制。 例如，大多数溶酶体水解酶， 遗传缺陷导致毁灭性的组织储存疾病， 糖基化酶，包括氨基己糖苷酶缺陷， 泰-萨二氏或山德霍夫病。此外，Brp 39是在一个亚组， 这些蛋白质不再显示催化活性，但在 各种各样的发育和分化过程， 通过结合几丁质低聚物的能力。 最后，这些结果是 生物技术相关性，如抗真菌药物的开发 剂.

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.