Binding of chloromethyl ketone substrate analogs to crystalline papain

Abstract

Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.

Mechanism of inhibition of papain by chicken egg white cystatin

“…According to the docking model, Gly-9 is in the close vicinity of but not in direct contact with the active site Cys-25 of papain. Within the preceding segment, assuming a tight-turn conformation ( fig.3), the side chain of Leu-8 could bind to the S2-subsite which mainly determines the substrate specificity of papain [17,18]. Our results obtained with N-terminally truncated forms of chicken cystatin strongly support this idea.…”

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

“…Thus the electrostatic modulation caused by the ionization of one or more acidic residues (electrostatic modulators) would be an important factor to weaken the Cys25 (Ϫ) …His159 (ϩ) interaction and permit the rotation of the His159 imidazolium ring from a more stable equiplanar conformation 32 to a conformation where the ND1-His159 atom is in the correct position to donate its proton to the nitrogen of the substrate peptide bond. 58 This analysis, which we will call the E SG3 ND1 attenuation hypothesis, gives support to the suggestion made by Pinitglang et al 23 : "It seems possible, however, that there may be a requirement to rearrange the ion pair geometry from that of an intimate ion pair to one in which the two components have separated to permit each to play its role in the rate-determining acylation process of the catalytic act. "…”

“…The electrostatic contribution from the basic residues is lesser in extent and in the sense to reinforce the electric field in the SG3 ND1 direction (⌬⌬V ϭ ⌬V mutated Ϫ ⌬V papain ϭ Ϫ1.003 kcal/mol). On the basis of these results and on the catalytic mechanism proposed by Drenth et al, 58 we formulate a possible explanation for the electrostatic switch mechanism of enzymatic regulation proposed by Pinitglang et al 23 The electrostatic contribution from the remote negatively charged residues would be important to attenuate the well-oriented local SG3 ND1 electric field, allowing the His159 to adopt a more favorable conformation to act as a proton donor in the acylation step of the enzymatic mechanism of papain. Thus the electrostatic modulation caused by the ionization of one or more acidic residues (electrostatic modulators) would be an important factor to weaken the Cys25 (Ϫ) …His159 (ϩ) interaction and permit the rotation of the His159 imidazolium ring from a more stable equiplanar conformation 32 to a conformation where the ND1-His159 atom is in the correct position to donate its proton to the nitrogen of the substrate peptide bond.…”

“…56,57 Before the minimization, the His159 CB-CG bond was rotated by ϳ30°in order to make the His159 ring, the Cys25 sulfur, and the Asn175-OD1 oxygen nearly equiplanar, in accordance with the catalytic mechanism proposed by Drenth et al 58 Water molecules were not considered in the refinement of the 1pop crystal structure but were retained for the refinement of the 9pap one. The 29 methanol molecules present in the 9pap crystal structure were substituted by water molecules taking the methanol oxygen atom as reference.…”

The picornaviral 3C proteinases: Cysteine nucleophiles in serine proteinase folds

“…Site-directed mutagenesis studies were suggestive but inconclusive (Ivanoff et al, 1986;Cheah et al, 1990;Hammerle et al, 1991;Kean et al, 1991). Speculation that a carboxylate third member was unnecessary to activate the more easily deprotonated thiol nucleophile, as generally accepted for the papain family of thiol proteinases (Drenth et al, 1976), has been (Fujinaga et al, 1987), and (C) papain (Drenth et al, 1976) generated using RASTER3D (Bacon & Anderson, 1988). Side chains of catalytic residues are color coded: nucleophiles (cysteine and serine) in yellow, general bases (histidines) in blue.…”