Mechanistic Enzymology of Phosphoryl Transfer Enzymes
磷酰基转移酶的机械酶学
基本信息
- 批准号:9253409
- 负责人:
- 金额:$ 29.92万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-09-15 至 2019-02-28
- 项目状态:已结题
- 来源:
- 关键词:AcidsActive SitesAddressAmino Acid SubstitutionBenchmarkingBiochemical ReactionBiologicalCatalysisCatalytic RNACell physiologyCharacteristicsChargeChemicalsChemistryChicagoCleaved cellClinicalCollaborationsDNADataDevelopmentDiagnosticElectrostaticsEnvironmentEnzymatic BiochemistryEnzyme Inhibitor DrugsEnzymesGeometryGoalsHealthHumanIndividualInvestigationIonsIsoenzymesIsotopesKineticsLightMeasuresMechanicsMetalsMethodsModelingMolecularMutationNucleotidesOligonucleotidesPancreatic ribonucleasePathogenesisPharmaceutical PreparationsProteinsProtonsRNARNA analysisReactionResearchSolventsStructureStudy modelsSystemTechnical ExpertiseTestingTherapeuticTimeTransferasebasecatalystcofactorcomputational chemistrydesigndrug developmentenzyme mechanismexperimental studyinhibitor/antagonistinsightnovelnucleobaseprotonationpublic health relevancequantumreaction ratesimulationtheoriestherapeutic enzymetherapeutic target
项目摘要
DESCRIPTION (provided by applicant): Understanding the mechanisms and transition states of phosphoryl transfer enzymes is important for understanding biological catalysis as well as facilitating the design of novel catalysts and supporting the development of enzyme inhibitors as potential drugs. In solution these reactions can occur by several different mechanisms with characteristic transition states. Transition state protonation, nucleophile and leaving group bonding and overall charge distribution are highly sensitive to the same catalytic modes (electrostatic stabilization, Bronsted acid/base, Lewis acid/base) that are proposed in models of catalysis by phosphoryl transferases. These insights strongly underscore the importance of addressing long standing unanswered questions in the field of biological catalysis: How do the catalytic modes employed by enzymes alter transition state charge distribution? Do enzymes with different active site geometries (isoenzymes) stabilize different transition states? Can differences in transition state structure facilitate the development of competitive inhibitors as potential drugs? Answering these questions will require a mechanistic framework grounded in theory and experiment for solution reactions, and the ability to apply this framework in structure-function studies to determine the transition states and catalytic modes for representative phosphoryl transfer enzymes. A powerful approach to understand enzyme mechanism is by analyzing kinetic isotope effects (KIEs), which measure the differences in ground state and transition state bonding, and integrating this information with molecular and quantum mechanical simulations to evaluate specific mechanistic scenarios and focus experimental efforts. Until now, technical barriers prohibited application of this powerful approach to reaction involving native RNA oligonucleotide substrates of ribozymes and protein phosphoryl transfer enzymes, leaving the questions highlighted above unanswered for an important enzyme class. Now, having established methods for KIE analyses RNA and nucleotide reactions, we using an integrated approach of theory and experiment to gain a comprehensive understanding of the mechanisms of phosphoryl transfer enzymes. The impact of these experiments is amplified by collaboration with Dr. Joseph Piccirilli (U Chicago) and Dr. Darrin York (Rutgers) who provide complementary technical strengths and importantly contribute independent intellectual perspectives. Our combined efforts are directed at providing new insights into how the active site environments of enzymes act to stabilize reaction transition states. The information gained will shed new light on the interplay between active site chemistry and chemical mechanism, which will significantly impact our understanding of biological catalysis and broadly support advances in design of new catalysts and discovery of inhibitors with potential therapeutic application.
描述(由申请人提供):了解磷酰基转移酶的机制和过渡态对于理解生物催化以及促进新型催化剂的设计和支持酶抑制剂作为潜在药物的开发非常重要。在溶液中,这些反应可以通过具有特征过渡态的几种不同机制发生。过渡态质子化,亲核体和离去基团键合和整体电荷分布是高度敏感的相同的催化模式(静电稳定,布朗斯台德酸/碱,刘易斯酸/碱),提出了由磷酰基转移酶的催化模型。这些见解强烈强调了解决生物催化领域长期悬而未决的问题的重要性:酶采用的催化模式如何改变过渡态电荷分布?具有不同活性部位几何形状的酶(同工酶)是否稳定不同的过渡态?过渡态结构的差异能否促进竞争性抑制剂作为潜在药物的发展?解决这些问题将需要一个基于理论和实验的解决方案反应的机制框架,并能够将此框架应用于结构-功能研究,以确定代表性磷酰基转移酶的过渡态和催化模式。 理解酶机制的一个强有力的方法是通过分析动力学同位素效应(KIE),它测量基态和过渡态键合的差异,并将这些信息与分子和量子力学模拟相结合,以评估特定的机制方案并集中实验工作。到目前为止,技术障碍阻止了这种强大的方法应用于涉及核酶和蛋白质磷酰基转移酶的天然RNA寡核苷酸底物的反应,使得上面强调的问题对于一个重要的酶类别没有答案。目前,我们已经建立了KIE分析RNA和核苷酸反应的方法,采用理论和实验相结合的方法,对磷酰基转移酶的作用机制有了全面的了解。这些实验的影响通过与Joseph Piccirilli博士(U芝加哥)和Darrin约克博士(罗格斯大学)的合作得到放大,他们提供互补的技术优势,并重要地贡献了独立的知识观点。我们的共同努力是针对提供新的见解如何酶的活性位点环境的作用,以稳定反应过渡态。所获得的信息将揭示活性位点化学和化学机制之间的相互作用,这将显着影响我们对生物催化的理解,并广泛支持新催化剂的设计和具有潜在治疗应用的抑制剂的发现。
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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MICHAEL E. HARRIS其他文献
MICHAEL E. HARRIS的其他文献
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{{ truncateString('MICHAEL E. HARRIS', 18)}}的其他基金
Specificity in Substrate Recognition and Catalysis by RNA Processing Enzymes
RNA 加工酶对底物识别和催化的特异性
- 批准号:
10190963 - 财政年份:2018
- 资助金额:
$ 29.92万 - 项目类别:
Specificity in Substrate Recognition and Catalysis by RNA Processing Enzymes
RNA 加工酶对底物识别和催化的特异性
- 批准号:
10434828 - 财政年份:2018
- 资助金额:
$ 29.92万 - 项目类别:
Mechanistic Enzymology of Phosphoryl Transfer Enzymes
磷酰基转移酶的机械酶学
- 批准号:
8697309 - 财政年份:2011
- 资助金额:
$ 29.92万 - 项目类别:
Mechanistic enzymology of phosphoryl transfer enzymes
磷酰基转移酶的机械酶学
- 批准号:
8329007 - 财政年份:2011
- 资助金额:
$ 29.92万 - 项目类别:
Mechanistic Enzymology of Phosphoryl Transfer Enzymes
磷酰基转移酶的机械酶学
- 批准号:
9105386 - 财政年份:2011
- 资助金额:
$ 29.92万 - 项目类别:
Mechanistic Enzymology of Phosphoryl Transfer Enzymes
磷酰基转移酶的机械酶学
- 批准号:
8909608 - 财政年份:2011
- 资助金额:
$ 29.92万 - 项目类别:
Mechanistic enzymology of phosphoryl transfer enzymes
磷酰基转移酶的机械酶学
- 批准号:
8184531 - 财政年份:2011
- 资助金额:
$ 29.92万 - 项目类别:
Determination of enzyme isotope effects by tandem ESI-Q/TOF mass spectrometry
通过串联 ESI-Q/TOF 质谱测定酶同位素效应
- 批准号:
7191481 - 财政年份:2007
- 资助金额:
$ 29.92万 - 项目类别:
Determination of enzyme isotope effects by tandem ESI-Q/TOF mass spectrometry
通过串联 ESI-Q/TOF 质谱测定酶同位素效应
- 批准号:
7345472 - 财政年份:2007
- 资助金额:
$ 29.92万 - 项目类别:
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