Abstract
Robust catalyst design requires clear understanding of the mechanisms by which molecular motions influence catalysis. This work investigates the connection between molecular motions and catalysis for the much debated enzyme Cyclophilin A (CypA) in wild-type (WT) form, and a variant that features a distal serine to threonine (S99T) mutation. Previous biophysical studies have proposed that conformational exchange between a ‘major’ active and a ‘minor’ inactive state on millisecond timescales plays a key role in catalysis. Here this hypothesis was addressed with molecular dynamics simulation techniques. The simulations reproduce well NMR-derived measurements of changes of activation barriers for the cis/trans amide group isomerization of a model substrate, and support X-ray crystallography derived evidence for a shift in populations of major and minor active site conformations between the wild-type and S99T mutant forms. Strikingly, exchange between major and minor active site conformations occurs at a rate that is 5 to 6 orders of magnitude faster than previously proposed. Further analyses indicate that the decreased catalytic activity of the S99T mutant is a result of weakened hydrogen bonding interactions between the substrate and several active site residues in the transition state ensemble. Weakened hydrogen bonding interactions in the S99T mutant are due to an overall increase in fast positional fluctuations of active site residues caused by poorer packing of sidechains. Therefore the previously described millisecond time scale coupled motions are not necessary to explain allosteric effects in the S99T Cyclophilin A mutant.