Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain

Abstract

In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.

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

“…The papain structural characteristics that are thought to give some conformational rigidity to the active site region are as follows: (1) the presence of a network of interface interactions between the two papain domains that makes large domain movements unlikely 7,40 ; and (2) the presence of important hydrogen-bonding interactions involving the side-chains of Gln19, Asn175, and Asp158. 38,39,41 Theoretical studies made by Rullmann et al 34 and Dijkman et al 32 show that the ion pair formation is very sensitive to the distance and the relative geometry of the Cys25 and His159 side chains.…”

“…The negatively charged Asp158 residue produces the more important contribution but only 28% of its total electric field intensity is aligned in the SG3 ND1 direction. Site-directed mutagenesis experiments made in papain 37,38 and caricain 21 showed that the Asp1583 Asn158 mutation (papain numbering) affects but does not reduce dramatically the catalytic activity of these enzymes (ϳ90% k cat /K m reduction). Noble et al 44 determined the pK a values of Asp158 as 2.8 and 2.0 in papain and caricain, respectively.…”

“…26 The factors that contribute to the formation and stabilization of the Cys25 (Ϫ) -His159 (ϩ) ion pair in the active site of cysteine proteinases are not completely understood and have been the object of many theoretical 29 -36,90,95 and experimental studies. [37][38][39][40][41] The results described in the literature indicate that one of the most important factors contributing to the stabilization of the active site ion pair is the overall enzyme electrostatic field. It is also suggested that the ion pair formation is very sensitive to the active site structure 38 and to the geometry of the Cys25 and His159 catalytic residues.…”

“…[37][38][39][40][41] The results described in the literature indicate that one of the most important factors contributing to the stabilization of the active site ion pair is the overall enzyme electrostatic field. It is also suggested that the ion pair formation is very sensitive to the active site structure 38 and to the geometry of the Cys25 and His159 catalytic residues. 34 An important aspect related to the regulation of the papain catalytic mechanism was described by Brocklehurst and coworkers.…”

Crystal structure of a caricain D158E mutant in complex with E‐64

“…These data were collected Site-directed mutants of papain at position 158 have been using beam line BL-6A2 equipped with a Weissenberg camera [26]. reported by M6nard et al [20,21]. In the first of these papers…”