CRYSTAL-STRUCTURE OF A PEPTIDYL PYRIDINIUM METHYL KETONE INHIBITOR WITH THROMBIN

The crystal structure of a complex between a bivalent peptidyl pyridinium methyl ketone inhibitor and human alpha-thrombin has been solved and refined at 2.0 Angstrom to an R factor of 0.18. The inhibitor, (D)cyclohexylalanine-Pro-Arg-(CH2N+C5H4CH2CO)-(Gly)(4)-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-cyclo-hexylalanine-(D)Glu (coded P596), which forms a reversible covalent complex with thrombin, is highly potent with a K-i = 4.6 +/- 1.0 x 10(-14) M, lower than that of recombinant hirudin. The N-terminal, activesite-directed portion of the inhibitor is linked to the fibrinogen recognition exosite binding portion by a tetraglycine segment. The strong electron-withdrawing effect provided by the permanent positive charge on the pyridinium nitrogen makes the arginyl carbonyl carbon more susceptible to nucleophilic attack. In the crystal, a covalent P596-thrombin complex is observed. The electron density surrounding the active site portion and the pyridinium of the inhibitor is very well defined, clearly showing the existence of a covalent bond between the Ser(195) O gamma and the now tetrahedral carbon of the inhibitor. The decreased binding ability of thrombin inhibitors containing N-terminal acetylation is discussed as is the effect of replacing the P-3 (D)phenylalanine with (D)cyclohexylalanine. The electron density surrounding the remainder of the inhibitor is generally well defined, the exceptions being the C-terminal (D)Glu, the highly flexible tetraglycine linker, and some of the solvent-directed side chains. The C-terminal part of the inhibitor corresponds to the exosite-directed inhibitor MDL-28050 [Qiu, X., Yin, M., Padmanabhan, K. P. Krstenansky, J. L., & Tulinsky, A. (1993) J. Biol. Chem. 268, 20318-20326], and the two bind to thrombin in a similar way.