期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 42, 页码 17974-17979出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0912190107
关键词
2D IR spectroscopy; enzyme dynamics
资金
- Roy J. Carver Charitable Trust
- National Science Foundation (NSF) [CHE-0644410, CHE-0715448]
- Nastional Institutes of Health [R01 GM65368]
- Center for Bioprocessing and Biocatalysis at the University of Iowa
The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzyme-catalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using two-dimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (N-3(-)). We observe that the frequency-frequency time correlation functions (FFCF) for the ternary complexes with NAD(+) and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window.
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