NEW INSIGHTS INTO ENZYME STRUCTURE AND FUNCTION
对酶结构和功能的新见解
基本信息
- 批准号:2181687
- 负责人:
- 金额:$ 18.34万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1989
- 资助国家:美国
- 起止时间:1989-07-01 至 1997-06-30
- 项目状态:已结题
- 来源:
- 关键词:Escherichia coli X ray crystallography active sites alkaline phosphatase chemical binding chemical substitution computer graphics /printing enzyme mechanism enzyme model enzyme structure enzyme substrate ionic bond metalloenzyme mutant nuclear magnetic resonance spectroscopy point mutation protein sequence protein structure function radiotracer site directed mutagenesis
项目摘要
In order to understand the molecular basis of diseases, particularly
those involving single amino acid substitutions, we need to elucidate the
relationship between protein structure and function at the molecular
level. Therefore, the long-term goals of this project are to acquire a
deeper understanding of the relationship between protein structure and
function by using alkaline phosphatase as a model system. The function
of this enzyme is to catalyze the nonspecific hydrolysis of phosphate
esters, and the lack of the activity of this enzyme results in the fatal
hereditary disease hypophosphatasia, which is due to insufficient
phosphate for bone calcification. Alkaline phosphatase from mammals is
closely related to the corresponding bacterial enzyme, and the enzyme
from Escherichia coli has become the model for the study of all alkaline
phosphatases. The specific aims of this proposal are to answer
fundamental questions concerning the relationship between the structure
and function of alkaline phosphatase. We will concentrate on the
molecular details of the catalytic mechanism, the need to maintain a
dimeric structure for the correct function of the enzyme, the mode by
which information is passed between the subunits, a molecular explanation
of intergenic complementation, the factors critical for correct secondary
structure formation, the function of the metals in catalysis, and the
contribution of the electrostatic field around the active site for
catalysis. In order to accomplish this goal, we will take advantage of
a set of thousands of point mutations created within the alkaline
phosphatase gene more than 30 years ago in an early effort to prove that
the gene and the protein produced from it were colinear. This set of
mutants today provides a unique resource for the investigation of the
interrelationship between the structure and function of alkaline
phosphatase. Analysis of this set of mutants as well as mutants created
during the last grant period will be accomplished by a variety of
techniques including x-ray crystallography, 31P and 113Cd NMR, 18(O)
isotope effects, and kinetic studies with phosphonates, phosphorothioates
and the chiral (Rp) [O16,O17, O18] p-nitrophenyl phosphate. Correlations
will be made between the functional changes induced by the amino acid
substitution and the three-dimensional structure of the mutant enzymes.
This work will not only be important for the understanding of this
particular system, but more importantly for formulating general concepts
about enzyme catalysis, and the function of metals in proteins, and
providing a molecular explanation for intergenic complementation.
为了了解疾病的分子基础,特别是
对于涉及单个氨基酸取代的那些,我们需要阐明
蛋白质结构与功能的关系
水平 因此,该项目的长期目标是获得
更深入地了解蛋白质结构与
功能通过使用碱性磷酸酶作为模型系统。 功能
该酶的作用是催化磷酸盐的非特异性水解
酯,缺乏这种酶的活性会导致致命的
遗传性疾病低磷酸酶,这是由于不足
磷酸盐用于骨骼钙化。 哺乳动物的碱性磷酸酶是
与相应的细菌酶密切相关,并且该酶
已成为研究所有碱性
磷酸酶 本提案的具体目的是回答
结构之间关系的基本问题
和碱性磷酸酶的功能。 我们将集中精力
分子细节的催化机制,需要保持一个
二聚体结构的酶的正确功能,模式,
在亚基之间传递的信息,
基因间互补的关键因素是正确的次生作用
结构的形成,金属在催化中的作用,
活性位点周围静电场的贡献,
催化作用 为了实现这一目标,我们将利用
一组数千个点突变产生的碱性
磷酸酶基因的早期努力证明,
该基因和由其产生的蛋白质共线。 这套
突变体今天提供了一个独特的资源调查的
碱金属结构与功能的相互关系
磷酸酶。 分析这组突变体以及产生的突变体
在最后一个赠款期间,将通过各种
技术,包括X射线晶体学,31 P和113 Cd NMR,18(O)
同位素效应,以及膦酸盐、硫代磷酸酯的动力学研究
和手性(Rp)[O 16,O 17,O 18]对硝基苯磷酸酯。 相关性
将在氨基酸诱导的功能变化之间进行
取代和突变酶的三维结构。
这项工作不仅对理解这一点很重要,
这是一个特定的系统,但更重要的是制定一般概念
关于酶催化,以及金属在蛋白质中的功能,
为基因间互补提供了分子解释。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('EVAN R KANTROWITZ', 18)}}的其他基金
DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE
直接观察底物引起的四元构象变化
- 批准号:
8362170 - 财政年份:2011
- 资助金额:
$ 18.34万 - 项目类别:
DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE
直接观察底物引起的四元构象变化
- 批准号:
8170121 - 财政年份:2010
- 资助金额:
$ 18.34万 - 项目类别:
DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE
直接观察底物引起的四元构象变化
- 批准号:
7954451 - 财政年份:2009
- 资助金额:
$ 18.34万 - 项目类别:
DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE
直接观察底物引起的四元构象变化
- 批准号:
7722147 - 财政年份:2008
- 资助金额:
$ 18.34万 - 项目类别:
TIME EVOLUTION OF THE ALLOSTERIC TRANSITION OF ASPARTATE TRANSCARBAMOYLASE
天冬氨酸转氨甲酰酶变构转变的时间演化
- 批准号:
7597962 - 财政年份:2007
- 资助金额:
$ 18.34万 - 项目类别:
TIME EVOLUTION OF THE ALLOSTERIC TRANSITION OF ASPARTATE TRANSCARBAMOYLASE
天冬氨酸转氨甲酰酶变构转变的时间演化
- 批准号:
7370443 - 财政年份:2006
- 资助金额:
$ 18.34万 - 项目类别:
TIME EVOLUTION OF THE ALLOSTERIC TRANSITION OF ASPARTATE TRANSCARBAMOYLASE
天冬氨酸转氨甲酰酶变构转变的时间演化
- 批准号:
7180422 - 财政年份:2005
- 资助金额:
$ 18.34万 - 项目类别:
STRUCTURE OF A COBALT-SUBSTITUTED MUTANT OF ALKALINE PHOSPHASE
碱性磷酸相的钴取代突变体的结构
- 批准号:
6972664 - 财政年份:2004
- 资助金额:
$ 18.34万 - 项目类别:
TIME EVOLUTION OF ALLOSTERIC TRANSITION OF ASPARTATE TRANSCARBAMOYLASE
天冬氨酸转氨甲酰酶变构转变的时间演化
- 批准号:
6976330 - 财政年份:2004
- 资助金额:
$ 18.34万 - 项目类别:
STRUCT & FUNCT OF MUTANT VERSIONS OF ALKALINE PHOSPHATASE FROM ESCHERICHIA COLI
结构体
- 批准号:
6221083 - 财政年份:1999
- 资助金额:
$ 18.34万 - 项目类别:
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