We propose to investigate the complex formation between trypsin and the substrate suc-AAPR-pNa using a protease inhibitor and cryo-trapping techniques. Features of the catalytic mechanisms of proteases have already been studied using cryogenic X-ray diffraction experiments, though recent development on instruments D19 and LADI III now allows cryogenic neutron diffraction studies to be performed. Using freeze trapping procedures neutron cryo-crystallography permits the identification of fast reaction intermediates, thus yielding structural information at a definite point in time. Cryogenic neutron experiments on enzyme-substrate complexes enable the investigation of residues protonation states in the active site of trypsin during substrate processing, as well as disclosing precise water molecule orientation and hydrogen bond network formation between substrate and enzyme. Further we will exploit the use of the newly developed carboloop mounting system for combined neutron and X-ray cryo-crystallography studies. In addition to the specific scientific goals of studying the trypsin enzymatic pathway,this work will also be of general value in helping develop new freeze-trapping protocol