(1) Dept. Crystallography, A. Mickiewicz Univ., Poznan, Poland
(2) Univ. of Gdansk, Poland
(3) Center for Biocrystallogr. Res., Pol. Acad. Sci.,
Poznan, Poland
maciej@tryptophan.ibch.poznan.pl
Several serious diseases, such as pulmonary emphysema, osteoporosis, and especially muscular dystrophy, are linked to abnormalities in the function of cysteine proteases. These enzymes, abundant in the living cells, are characterized by a catalytic triad, C-H-N, the thiol group of which is the reactive nucleophile. There is a variety of natural inhibitors of cysteine proteases, ranging from sizable proteins (cystatin superfamily) to relatively small molecules, such as E-64. The latter compound, which is among the most potent inhibitors, contains an oxirane ring and forms a covalent bond with the essential SH group. We have determined the crystal structure of a covalent complex between the 212-amino-acid long cysteine proteinase papain, and an irreversible oxirane-based inhibitor. The inhibitor, Z-RLV-"psi"[CH2-NH]-CO-[oxirane]-CO-Ph, contains a tripeptide fragment connected to the -CO-[oxirane]-CO- moiety, which is also present (but in carboxylate form) in E-64. The complex was prepared by treating fully active papain with excess of aqueous solution of the inhibitor until no enzymatic activity could be detected. The complex was crystallized at 19 deg. C from 1.5% protein solution with methanol/ ethanol as the precipitant, using a modification of the procedure described by Kim em et al.[1]. Plate-like crystals appeared after 3-4 days. They are monoclinic, P21, with a=42.0, b=50.0, c=55.0 A, beta=95.9 deg, contain one complex molecule in the asymmetric unit, and correspond to the historically first papain form crystallized by Drenth [2]. The crystals are stable in the X-ray beam. Intensity data extending to 1.9- A resolution were collected for a crystal measuring 0.3x0.2x0.03 mm. They are 92.2% complete and scale with Rsym=0.097. The structure was solved by molecular replacement using the {apo} form of papain [3] as a model (c.c.=0.69, R=0.51). Preliminary electron density maps clearly showed the inhibitor which is covalently linked to the active-site Cys-25 of the enzyme. The inhibitor moiety has been built into the electron density and the model, together with several water and methanol molecules, is currently being refined (R=0.21).
1. M.-J. Kim et al. (1992). Biochem J. {287}, 797.
2. J. Drenth (1959), Nature {184}, 1718.
3. I.G. Kamphius et al. (1987), J. Mol. Biol. {179}, 233.