Crystal structure of a papain-E-64 complex

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Abstract
E-64 [1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-guanidinobutane] is an irreversible inhibitor of many cysteine proteases. A papain-E-64 complex was crystallized at pH 6.3 by using the hanging drop method. Three different crystal forms grew in 3-7 days; the form chosen for sturcture analysis has space group P212121, with a = 42.91 (4) .ANG., b = 102.02(6) .ANG., c = 49.73 (2) .ANG., and Z = 4. Diffraction data were measured to 2.4-.ANG. resolution, giving 9367 unique reflections. The papain structure was solved by use of the molecular replacement method, and then the inhibitor was located from a difference electron density map and fitted with the aid of PS330 computer graphics system. The structure of the complex was refined to R = 23.3%. Our analysis shown that a covalent link is formed between the sulfur of the active-site cysteine 25 and the C-2 atom of the inhibitor. Contrary to earlier predictions, the E-64 inhibitor clearly interacts with the S subsets on the enzyme rather than the S'' subsites,and papain''s histidine 159 imidazole group plays a binding rather than a catalytic role in the inactivation process.