One of the ways we know about the mechanism of enzymes is through the use of xray crystallography structures of trapped intermediates or inhibitors bound to enzymes. In a paper by Radisky and Koshland[1], an acyl intermediate of trypsin (PDB code 2AGG was characterized.
Serine proteases use a covalent mechanism to catalyze the hydrolysis of a peptide bond. A covalent bond is formed between and a substrate . The between one of the Ser O configurations and the alpha carbon of the substrate lysine is the distance of a C-O bond, indicating that the covalent intermediate is indeed formed. There is a situated in the active site, primed to cleave the acyl intermediate by the same that participates in the catalytic triad.
Specificity of the proteases is determined by a binding pocket. Trypsin is specific for large, basic amino acids; its binding pocket contains an residue at the base of the pocket.
