Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin with high affinity. In the trypsin-BPTI complex the reactive peptide sequence of BPTI is positioned ideally for peptide bond hydrolysis. However, the peptide bond hydrolysis is similar to 10(11) slower for BPTI than for peptide substrates. Wr have applied the quantum mechanical-free energy (QM-FE) approach and molecular dynamics simulations to investigate peptide bond cleavage in the trypsin-BPTI and the trypsin-substrate systems. The activation free energy calculated for BPTI as substrate suggests a decrease in catalytic rate by 10(6)-10(9) ill qualitative agreement with the similar to 10(11) found experimentally. We provide evidence to support the hypothesis that the remaining 10(2)-10(5) decrease in rate arises from the fact that the peptide bond in BPTI. once cleaved, is much more likely to re-ligate than to be further hydrolyzed to form products which will be released from trypsin. We have also demonstrated that the QM-FE approach, in combination with the free energy component analysis, should be a useful method to investigate enzyme catalysis and to derive information on the roles of different enzyme groups on the reaction energies,.