AUTOPROTEOLYSIS OF N-TERMINAL NUCLEOPHILE HYDROLASE

描述(摘自摘要)：溶酶体糖基天冬酰胺酶水解物 在天冬氨酸连接的糖蛋白中连接碳水化合物和蛋白质的酰胺键。 该酶加入蛋白酶体(Lowe等人，1995；Groll等人，1997)和 青霉素酰化酶(Duggleby等人，1995)作为一类酰胺酶 催化使用经过处理的N-末端苏氨酸或丝氨酸作为 极化碱基和亲核剂。这种酶另一个耐人寻味的方面是 单链前体通过分子内自蛋白分解来处理 产生保守的N-末端苏氨酸和活性酰胺酶(Guan等人， 1996年)。有了晶体，郭博士建议确定晶体 糖基天冬酰胺酶前体(酶原)的结构研究 这种分子内自蛋白分解的机制，以及激活过程 酶活性。基于构造几何和进化守恒 序列中，看起来对自身蛋白分解很重要的残基将是 被选中进行定点突变。突变的酶随后将受到 通过圆二色谱(CD)进行动力学分析和/或进一步的物理研究 结晶学。郭博士将进一步研究二聚化在 分子内的自蛋白分解。他还使变种人具体化 活性降低的成熟(自切)形式的蛋白质。通过应用 冰晶学技术，他将试图稳定 用于X射线结构测定的酶-底物(或抑制剂)复合体 允许对酶机制进行更详细的检查。酶制剂 活性和酶的激活是许多生理学的中心。郭医生的 这项工作解决了原子分辨率下的功能机制，并且应该 有助于加深对基本生物过程的理解。 这项研究计划的广泛、长期目标是了解 酶机制的分子基础，以及蛋白质剪接。这些工具 使用的是x射线结晶学、CD、分子生物学和蛋白质。 化学反应。

DESCRIPTION (Adapted from abstract): Lysosomal glycosylasparaginase hydrolyzes the amide bond joining carbohydrate to protein in Asn-linked glycoproteins. This enzyme joins the proteasome (Lowe et al., 1995; Groll et al., 1997) and penicillin acylase (Duggleby et al., 1995) as a class of amidases that catalytically use a processed N-terminal threonine or serine as both a polarizing base and a nucleophile. Another intriguing aspect of this enzyme is that a single chain precursor is processed by intramolecular autoproteolysis to yield the conserved N-terminal threonine and an active amidase (Guan et al., 1996). With crystals in hand, Dr. Guo proposes to determine the crystal structures of glycosylasparaginase precursor (proenzyme) to elucidate the mechanism of this intramolecular autoproteolysis, and activation process of enzyme activity. Based on structural geometry and evolutionarily conserved sequence, the residues appearing to be important for autoproteolysis will be selected for site-directed mutagenesis. Mutant enzymes will then be subject to kinetic analysis, and/or further physical studies by circular dichroism (CD) or crystallography. Dr Guo will further investigate the role of dimerization in the intramolecular autoproteolysis. He has also crystallized the mutant proteins of reduced activity in their mature (autocleaved) form. By applying cryocrystallography techniques, he will attempt to stabilize the enzyme-substrate (or inhibitor) complexes for x-ray structure determination, to allow a more detailed examination of the enzymatic mechanism. The enzymatic activities and enzyme activation are central to much of physiology. Dr. Guo's work addresses the mechanisms of function at atomic resolution, and should contribute to an increased understanding of the essential biological processes. The broad, long-term objectives of this research plan are to understand the molecular basis of enzymatic mechanisms, as well as protein splicing. The tools employed are x-ray crystallography, CD, molecular biology, and protein chemistry.