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。我们会 专门研究水解甲壳素和甲壳素的糖基水解酶- 像聚合物一样变成更小的糖，最终变成单糖。 这是由一组酶执行的，这些酶要么在中间裂解 或通过释放单糖或双糖 多糖的非还原或还原末端及其 碎片。为了实现我们的目标，我们计划使用 分子与结构相结合的计算化学方法 研究几丁质酶A、Brp39(乳房退化)的生物技术 蛋白质)和几丁酶，都属于同一几丁质分解家族的成员。 例如，将使用分子力学/动力学来研究 量子偶联时几丁质酶A与多糖的结合 将用力学/分子力学来研究催化剂。 反应机理。分子生物学和生物化学将被用于 验证计算结果并生产几丁质酶A 与多糖结合而不切割的突变体。一个三维的 失活几丁质酶A结合突变体的晶体结构 将通过X射线结晶学来确定底物。因此， 综合计算、分子生物学和结构结果 这一努力将产生一幅清晰详细的分子图景 了解负责绑定的各种相互作用 几丁质酶A的多糖和几丁质酶A的明确图片 催化反应机理。类似的程序将在 Brp39和Chitobiase的研究。从这项工作中获得的新知识 将适用于50多种糖基水解酶中的几种 家人。这是因为存在着对生命至关重要的 催化和其他活性中心残基；2)共同的结构演化 具有(β/α)8桶折叠基序的糖苷酶；和3] 类似的酸/碱或底物辅助催化反应机理。 这些结果对于理解溶酶体的存储具有医学意义。 疾病机制。例如，大多数溶酶体水解酶的 基因缺陷会导致毁灭性的组织储存疾病 糖苷酶，包括己糖氨酸酶缺陷 泰-萨克斯病或桑德霍夫病。此外，Brp39属于 这些蛋白质不再表现出催化活性，但在 很可能是各种发育和分化过程 通过结合甲壳素的低聚体的能力。最后，这些结果是 与生物技术相关的，例如在抗真菌药物的开发中 探员们。

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.