The precise catalytic mechanism of peptidyl-prolyl cis-trans isomerases (PPIases) has been elusive, despite many experimental and computational studies. The more than 5 orders of magnitude speedup achieved in catalysis by cyclophilin A (CypA) has been attributed to several factors, including substrate desolvation, enzyme dynamics, and preferential binding of the transition state. Here, we explore the conformational space of a substrate analogue of CypA using accelerated molecular dynamics, free in solution and in the active site of CypA, in order to probe its conformational interconversion during catalysis. We show that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. Our results suggest that the loss in conformational entropy at the transition state relative to the cis and trans states in the free substrate is decreased in the complex. This relative change in conformational entropy contributes favorable to the free energy of stabilizing the transition state by CypA. We also show that the ensuing intramolecular polarization, as a result of the loss in pseudo double bond character of the peptide bond at the transition state, contributes only about -1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. Our results provide further insights into the mechanism of CypA, a member of a poorly understood family of enzymes that are central to many biological processes.