# Ultrafast dynamics in enzyme catalysis

> **NIH NIH R35** · SOUTHERN ILLINOIS UNIVERSITY CARBONDALE · 2020 · $328,145

## Abstract

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 experime...

## Key facts

- **NIH application ID:** 9948683
- **Project number:** 5R35GM119818-05
- **Recipient organization:** SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
- **Principal Investigator:** Sean D Moran
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $328,145
- **Award type:** 5
- **Project period:** 2016-09-07 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9948683

## Citation

> US National Institutes of Health, RePORTER application 9948683, Ultrafast dynamics in enzyme catalysis (5R35GM119818-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9948683. Licensed CC0.

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