Date of Degree
PhD (Doctor of Philosophy)
Christopher M. Cheatum
Femtosecond to picosecond dynamics of enzyme active sites is relatively unknown. With the advent of two-dimension infrared spectroscopy, it is now possible to characterize thermally activated motions at this time scale in functionally relevant enzyme complexes. We present a series of measurements on complexes of formate dehydrogenase (FDH) using transitional-state analog inhibitor azide as a reporter. Our results show that for ternary complexes with NAD+ and NADH, the frequency-frequency correlation function (FFCF) decays completely. This indicates that the active site at the transitional state is rigid and the azide completely samples the high frequency fluctuations within a couple of picoseconds. In contrast, the FFCF of binary complexes show significant static component to the overall decay. Such functionally relevant dynamics observed in FDH is difficult to investigate in other enzymatic system due to the lack of suitable probes. We have synthesized two new mid-IR active analogs of NAD+ , azido-NAD+ and picolyl azido adenine dinucleotide (PAAD), each of which have the potential to be a general probe for enzyme dynamics. These new probes were fully characterized as potential candidates for probing the active sites of NAD-dependent enzymes. As with FDH-azide complex, PAAD bound to FDH shows a significant static component. Our result with ternary complex of FDH with PAAD and azide shows that the active site of this enzyme still represents the transitional state of the system albeit with perturbations and both the reporter completely samples the fluctuations in the active site within 10 ps. This results indicate that the active site is rigid within the immediate vicinity of the probes and that this rigidity might be a general characteristic of the entire active site at the transitional state.
Copyright 2010 Samrat Dutta
Dutta, Samrat. "Developing new probes of functionally relevant dynamics in NAD-dependent enzymes." dissertation, University of Iowa, 2010.