X-ray structures of trypsin from bovine pancreas inactivated by diphenyl [N-(benzyloxycarbonyl)amino](4-amidinophenyl)methanephosphonate [Z-(4-AmPhGly) P (OPh) 2 ] were determined at 113 and 293 K to 1.8 Å resolution and refined to R factors of 0.211 (113 K) and 0.178 (293 K). The structures reveal a tetrahedral phosphorus covalently bonded to the Oγ of the active site serine. Covalent bond formation is accompanied by the loss of both phenoxy groups. The D-stereoisomer of Z-(4-AmPhGly) P -(OPh)

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... 2 is not observed in the complex. The L-stereoisomer of the inhibitor forms contacts with several residues in the trypsin active site. One of the phosphonate oxygens is inserted into the oxyanion hole and forms hydrogen bonds to the amides of Gly193, Asp194, and Ser195. The second phosphonate oxygen forms hydrogen bonds to N 2 of His 57. The p-amidinophenylglycine moiety binds into the trypsin primary specificity pocket, interacting with Asp189.