This work investigates the connection between stochastic protein dynamics and function for the enzyme cyclophilin A (CypA) in wild-type form, and three variants that feature several mutations distal from the active site. Previous biophysical studies have suggested that conformational exchange between a ‘major’ active and a ‘minor’ inactive state on millisecond timescales plays a key role in catalysis for CypA. Here this hypothesis is addressed by a variety of molecular dynamics simulation techniques. Strikingly we show that exchange between major and minor active site conformations occurs at a rate that is 5 to 6 orders of magnitude faster than previously proposed. The minor active site conformation is found to be catalytically impaired, and decreased catalytic activity of the mutants is caused by changes in Phe113 motions on a ns-μs timescale. Therefore millisecond timescale motions may not be necessary to explain allosteric effects in cyclophilins.