Thiocyanate probes have been introduced at site-specific positions in ketosteroid isomerase (KSI) and dihydrofolate reductase (DHFR). A mixed quantum mechanical/molecular mechanical (QM/MM) approach was developed for calculating the infrared spectra of these nitrile groups. The QM region is treated with a reparameterized semiempirical method, and the nitrile stretching frequency is calculated with a grid-based method as the splitting between vibrational states. This approach was used to investigate the impact of intermediate analogue binding on the vibrational frequencies of nitrile probes in KSI. The calculations reproduce the experimental data and provide atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by these probes. This approach is being used to calculate the vibrational frequencies of nitrile probes in DHFR for intermediates along the catalytic cycle, enabling the investigation of changes in hydrogen bonding, electrostatics, and active site reorganization along the catalytic reaction pathway.

Calculation of vibrational shifts of nitrile probes in enzymes: Electrostatics, hydrogen bonding, and reorganization

246th National Meeting (2013)

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