Ja. Bertrand et al., INHIBITION OF TRYPSIN AND THROMBIN BY AMINO(4-AMIDINOPHENYL)METHANEPHOSPHONATE DIPHENYL ESTER DERIVATIVES - X-RAY STRUCTURES AND MOLECULAR-MODELS, Biochemistry, 35(10), 1996, pp. 3147-3155
X-ray structures of trypsin from bovine pancreas inactivated by diphen
yl [N-(benzyloxycarbonyl)amino] (4-amidinophenyl)methanephosphonate [Z
-(4-AmPhGly)(P)(OPh)(2)] were determined at 113 and 293 K to 1.8 Angst
rom resolution and refined to R factors of 0.211 (113 K) and 0.178 (29
3 K). The Structures reveal a tetrahedral phosphorus covalently bonded
to the O gamma 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)(2) is not observed in the complex. The L-stereo
isomer of the inhibitor forms contacts with several residues in the tr
ypsin active site. One of the phosphonate oxygens is inserted into the
oxyanion hole and forms hydrogen bonds to the amides of Gly193, Asp19
4, and Ser195. The second phosphonate oxygen forms hydrogen bonds to N
epsilon 2 of His 57. The p-amidinophenylglycine moiety binds into the
trypsin primary specificity pocket, interacting with Asp189. The amid
e forms a hydrogen bond to the carbonyl oxygen atom of Ser214. The inh
ibitor moiety, from the 113 K structure of trypsin inactivated by the
reaction product of Z-(4-AmPhGly)(P)(OPh)(2), was docked into human th
rombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., & Ho
fsteenge, J. (1989) EMBO J. 8, 3467-3475] and energy minimized. The in
hibitor fits well into the thrombin active site, forming favorable con
tacts similar to those in the trypsin complex with no bad contacts.