Ultrafast dynamics in enzyme catalysis

酶催化中的超快动力学

• 批准号: 9142918
• 负责人: Sean D Moran
• 金额: $ 28.95万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-07 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9142918
• 关键词: Active Sites; Amino Acid Sequence; Biological; Biological Process; Biological Sciences; Catalysis; Communication; Coupled; Drug Design; Electronics; Energy Transfer; Enzymes; Frequencies; Future; Goals; Isotopes; Label; Lead; Link; Measurement; Measures; Methods; Motion; Mutagenesis; Pathway interactions; Peptide Sequence Determination; Process; Property; Protein Biosynthesis; Protein Chemistry; Proteins; Proxy; Pump; Reaction; Relaxation; Research; Role; Scaffolding Protein; Spectrum Analysis; System; Techniques; Testing; Time; Variant; analog; base; catalyst; design; disease-causing mutation; electric field; enzyme activity; enzyme model; human disease; metabolic abnormality assessment; millisecond; mutant; programs; research study; scaffold; tool; trend

PROJECT SUMMARY Enzyme dynamics span a broad range of timescales, from milliseconds and microseconds down to picoseconds and femtoseconds. Catalysis of biological reactions is linked to these motions, but the role of ultrafast dynamics in the femtosecond and picosecond range remains controversial. Because diverse fields in the biological sciences, such as the study of metabolism and drug design, depend on a detailed understanding of enzyme function, resolving this controversy could have important implications for our understanding of many human diseases. We approach this problem using a combination of traditional protein chemistry techniques and 2D IR spectroscopy to correlate dynamics measurements with biologically-relevant function. In this proposal, we describe our current and future efforts to characterize differences in the ultrafast motions of enzymes in sets of mutants with different degrees of activity. The long-term goal of this project is to discover how protein sequence variations, including disease-causing mutations, can influence the catalytic function of enzymes via motions on a similar timescale as bond formation and breaking in an enzyme’s active site. Our direct objectives in this research are to develop the experimental tools needed to observe communication between an enzyme’s active site and scaffold, both via mutagenesis and spectroscopy. We hypothesize that trends in the activities of mutants will also be reflected in their ultrafast dynamics due to modulation of barrier crossing during reactions. We will use protein synthesis and labeling techniques to prepare a set of model enzymes with active site vibrational labels, and will use 2D IR spectroscopy to characterize local electric field dynamics. We will utilize vibrationally-labeled substrate analogs as well as protein-based labels in concert with variations in pump-rpbe waiting time to characterize these dynamics. Using random mutagenesis and activity screens, we will identify mutants of these enzymes with altered catalytic rates. Dynamics measurements of active site labels will be correlated to enzyme activities and other properties such as fold stability. Our research program also includes experiments designed to measure non-equilibrium dynamics that may influence catalysis. We will also use our labeled systems to investigate the origins of non-exchangeable heavy isotope effects on catalytic efficiency. Later, we will examine the efficiency of energy transfer within the active site, and between the active site and scaffold, using variations on 2D IR spectroscopy. We will use dual- frequency 2D IR spectroscopy to probe pairs of labels for evidence of vibrational energy transfer pathways. Finally, we will use transient 2D IR spectroscopy to examine pathways for energy relaxation through the protein scaffold after electronic photoexcitation of active site moieties, which we use as a proxy for relaxation after a reaction step. In all cases, we will perform these experiments against mutant backgrounds in which catalysis is altered.

项目总结