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MECHANISMS OF ENZYMIC AND HYDRIDE TRANSFERS

酶和氢化物转移的机制

基本信息

批准号：
7093614
负责人：
GREGORY A PETSKO
金额：
$ 37.75万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-01-16 至 2008-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7093614
关键词：
X ray crystallography acidity /alkalinity active sites catalyst chemical binding chemical kinetics cofactor computer simulation dihydrofolate reductase enzyme mechanism enzyme structure enzyme substrate hydrogen bond ionic bond isomerase oxidoreductase site directed mutagenesis xylose

项目摘要

DESCRIPTION (provided by applicant): The objective of this project is to use X-ray crystallography, computer simulations, site-directed mutagenesis and kinetic measurements to determine what structural features in an enzyme active site promote efficient catalysis of hydrogen ion transfer. Transfer of a hydrogen, usually in the form of either a proton or a hydride ion is the most common chemical reaction in biology, and is also perhaps the simplest, yet it is not always an easy reaction to carry out rapidly. When the donor and acceptor atoms are unactivated carbons or oxygens, the uncatalyzed reactions are very slow, because the -C-H and -O-H groups are not very acidic (both typically have pKa values near 20). Yet enzymes are able to accelerate the transfer of H+ and H- from -C-H and -O-H by more than 15 orders of magnitude in some cases, achieving turnover numbers exceeding 1000 per second. Establishing how enzymes activate substrates, cofactors and their own catalytic groups to effect such catalysis is the goal of this project. We have selected several model systems for study. For proton transfer catalysis: mutarotase (GalM), which catalyses both ring opening and proton transfer; and ketosteroid isomerase (KSI), which utilizes a prototypical acid/base mechanism. For hydride transfer: xylose isomerase (XyI), which catalyzes sugar ring opening followed by metal-mediated 1,2-hydride transfer and dihydrofolate reductase (DHFR), where the hydride donor is NAD. For mutarotase and KSI our aim is to understand how the enzymes increase the basicity of the catalytic base and lower the pKa of the carbon acid and how the transition states are stabilized. For XyI, we aim to learn how the two metal ions in the active site cooperate to promote hydride transfer and how the ring-opening reaction is catalyzed. For DHFR we are studying the orientation and distance requirements for hydride donor and substrate positioning, and the possible role of coenzyme and substrate strain. The protic environment in the active sites and the role of protein dynamics in catalysis are also being probed for all these enzymes computationally and by ultra-high resolution (beyond approximately IA resolution) X-ray diffraction.

描述（由申请人提供）：该项目的目标是利用 X 射线晶体学、计算机模拟、定点诱变和动力学测量来确定酶活性位点的哪些结构特征促进氢离子转移的有效催化。氢的转移（通常以质子或氢负离子的形式）是生物学中最常见的化学反应，也可能是最简单的，但它并不总是一个容易快速进行的反应。当供体和受体原子是未活化的碳或氧时，非催化反应非常慢，因为-C-H和-O-H基团的酸性不是很强（两者的pKa值通常接近20）。然而，在某些情况下，酶能够将 H+ 和 H- 从 -C-H 和 -O-H 的转移加速超过 15 个数量级，实现每秒超过 1000 个的转换次数。该项目的目标是确定酶如何激活底物、辅因子及其自身的催化基团以实现这种催化作用。我们选择了几个模型系统进行研究。用于质子转移催化：变旋酶（GalM），其催化开环和质子转移；和酮类固醇异构酶（KSI），它利用典型的酸/碱机制。对于氢化物转移：木糖异构酶 (Xyl)，其催化糖环开环，然后进行金属介导的 1,2-氢化物转移；以及二氢叶酸还原酶 (DHFR)，其中氢化物供体是 NAD。对于变旋酶和 KSI，我们的目标是了解酶如何增加催化碱的碱性并降低碳酸的 pKa 以及如何稳定过渡态。对于XyI，我们的目标是了解活性位点中的两种金属离子如何合作促进氢化物转移以及如何催化开环反应。对于 DHFR，我们正在研究氢化物供体和底物定位的方向和距离要求，以及辅酶和底物菌株的可能作用。所有这些酶的活性位点质子环境以及蛋白质动力学在催化中的作用也正在通过计算和超高分辨率（超过约 IA 分辨率）X 射线衍射进行探测。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Probing the catalytic mechanism of yeast triose phosphate isomerase by site-specific mutagenesis.

通过定点诱变探讨酵母磷酸丙糖异构酶的催化机制。

DOI：
10.1042/bst0120229
发表时间：
1984
期刊：
Biochemical Society transactions
影响因子：
3.9
作者：
Petsko,GA;DavenportJr,RC;Frankel,D;RaiBhandary,UL
通讯作者：
RaiBhandary,UL

A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase.

DOI：
10.1021/bi201089v
发表时间：
2011-10
期刊：
Biochemistry
影响因子：
2.9
作者：
Srinivas Somarowthu;H. Brodkin;J. A. D'aquino;D. Ringe;M. Ondrechen;P. Beuning
通讯作者：
Srinivas Somarowthu;H. Brodkin;J. A. D'aquino;D. Ringe;M. Ondrechen;P. Beuning

Observation of unstable species in enzyme-catalyzed transformations using protein crystallography.

DOI：
10.1016/s1367-5931(99)00057-5
发表时间：
2000-02
期刊：
Current opinion in chemical biology
影响因子：
7.8
作者：
G. Petsko;D. Ringe
通讯作者：
G. Petsko;D. Ringe

Time-of-flight neutron diffraction study of bovine γ-chymotrypsin at the Protein Crystallography Station.

在蛋白质晶体学站对牛γ-胰凝乳蛋白酶进行飞行时间中子衍射研究。

DOI：
10.1107/s1744309111009341
发表时间：
2011
期刊：
Acta crystallographica. Section F, Structural biology and crystallization communications
影响因子：
0
作者：
Lazar,LouisM;Fisher,SZoe;Moulin,AaronG;Kovalevsky,Andrey;Novak,WalterRP;Langan,Paul;Petsko,GregoryA;Ringe,Dagmar
通讯作者：
Ringe,Dagmar

Inhibition of chymotrypsin by a complex of ortho-vanadate and benzohydroxamic acid: structure of the inert complex and its mechanistic interpretation.