Multiple binding of inhibitors in the complex formed by bovine trypsin and fragments of a synthetic inhibitor, 4-[4-(N,N- dimethylcarbamoxylmethoxycarbonylmethyl)phenoxycarbonylphenyl+ ++] guanidinium methanesulfonate (FOY-305).

The crystal of bovine trypsin complexed with a potent inhibitor, 4-[4-(N,N- dimethylcarbamoylmethoxycarbonylmethyl)phenoxycarbonylphenyl ]guanidinium (FOY-305) in the novel orthorhombic from with a low molecular packing density was studied by the X-ray diffraction method. Using synchrotron radiation, the intensity data were collected to 1.8 A resolution. The structure was solved by molecular replacement methods, and refined to an R-factor = 18.0% for 14364 reflections by the restrained least-squares method. The final difference Fourier maps revealed that hydrolyzed inhibitor fragments bind with the protein at multiple sites around the active center of trypsin. The structural feature in the crystalline state probably corresponds to a statistical average of several complexes which would be formed between the inhibitor and trypsin during the binding and releasing process in solution.