RUI: Comparative Structure/Function Analysis of Phosphagen Kinases

磷酸激酶（PK）是迄今为止在所有动物中发现的高度保守的酶。它们通过在高活动期间维持稳定的ATP浓度在能量稳态中发挥关键作用。肌酸激酶（CK）是在脊椎动物中发现的唯一PK，而精氨酸激酶（AK）在无脊椎动物中发现。与CK不同，AK的天然状态是单体的，除了在少数情况下。四级结构差异的原因目前尚不清楚，但可能是基于生理功能或稳定性的不同要求。本研究的总体目标是利用比较生物化学的方法，以扩大目前的理解如何结构决定功能的PKs。这些项目特别关注PKs中底物结合、亚基间协同性和双分子反应机制之间的相互作用。PKs为这些问题的研究提供了一些独特的优势，因为它们沿着沿着不同的轨道进化，但仍然执行相同的基本生理功能。本研究的具体目标是：1）研究亚基间通讯的氨基酸决定簇（负协同性）; 2）确定足以产生马蹄蟹AK的功能性同源二聚体形式的最小残基集; 3）确定小说的结构和功能，来自革兰氏阳性菌的推定PK，其似乎缺少胍基底物特异性所必需的结构组分; 4）确定来自秀丽隐杆线虫的五种新的推定AK的结构和功能，作为为将这些研究置于生理背景下的未来实验建立基础的手段。这些目标的完成将提供一个了解AK和CK之间的一级结构的差异如何引起观察到的四级结构的差异，亚基协同性，底物特异性和催化磷酸盐转移在这个重要的和广泛的蛋白质家族。广泛的影响：伍斯特学院以其独立学习计划而闻名全国。这项研究将加强对大量有潜力的年轻科学家进行现代生物化学和分子生物学技术方面的出色科学培训。此外，这些项目还为生物学、化学和生物化学/分子生物学的本科生提供了合作研究的模式。

Phosphagen kinases (PKs) are highly conserved enzymes found in all animals examined to date. They play a key role in energy homeostasis by maintaining stable ATP concentrations during times of high activity. Creatine kinase (CK) is the exclusive PK found in vertebrates, whereas arginine kinase (AK) is found in invertebrates. Unlike CK, the native state of AK is monomeric, except in a few cases. The reasons for this difference in quaternary structure is currently unknown, but may be based in differing requirements of physiological function or stability. The overall goal of this research is to utilize a comparative biochemical approach to extend current understanding of how structure determines function in PKs. These projects specifically focus on the interplay between substrate binding, inter-subunit cooperativity and bimolecular reaction mechanisms in PKs. PKs offer some unique advantages to the study of these issues given that they have evolved along different tracks, yet still perform the same basic physiological function. The specific goals of this research are to 1) investigate the amino acid determinants of inter-subunit communication (negative cooperativity) observed in rabbit muscle CK; 2) determine the minimal set of residues sufficient to generate a functional homodimeric form of horseshoe crab AK; 3) determine the structure and function of novel, putative PKs from Gram positive bacteria that appear to be missing the structural components necessary for guanidino-substrate specificity; 4) determine the structure and function of five, novel, putative AKs from Caenorhabditis elegans as a means of establishing a foundation for future experiments for placing these studies within a physiological context. Completion of these goals will provide an understanding of how the differences in primary structure between AK and CK give rise to the observed differences in quaternary structure, subunit cooperativity, substrate specificity and catalysis of phosphate transfer within this important and widespread protein family. Broad Impact: The College of Wooster has a national reputation for its Independent Study program. Excellent scientific training in modern biochemical and molecular biology techniques for a significant number of young potential scientists will be enhanced by this research. In addition, these projects serve as a model of collaborative research for undergraduate students in biology, chemistry and biochemistry / molecular biology.