Structural and Functional Roles of Asparagine 175 in the Cysteine Protease Papain

Abstract

The role of the asparagine residue in the Cys-His-Asn "catalytic triad" of cysteine proteases has been investigated by replacing Asn175 in papain by alanine and glutamine using site-directed mutagenesis. The mutants were expressed in yeast and kinetic parameters determined against the substrate carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)- L-arginine. At the optimal pH of 6.5, the specificity constant (k(cat)/KM)obs was reduced by factors of 3.4 and 150 for the Asn175-->Gln and Asn175-->Ala mutants, respectively. Most of this effect was the result of a decrease in k(cat), as neither mutation significantly affected KM. Substrate hydrolysis by these mutants is still much faster than the non-catalytic rate, and therefore Asn175 cannot be considered as an essential catalytic residue in the cysteine protease papain. Detailed analyses of the pH activity profiles for both mutants allow the evaluation of the role of the Asn175 side chain on the stability of the active site ion pair and on the intrinsic activity of the enzyme. Alteration of the side chain at position 175 was also found to increase aggregation and proteolytic susceptibility of the proenzyme and to affect the thermal stability of the mature enzyme, reflecting a contribution of the asparagine residue to the structural integrity of papain. The strict conservation of Asn175 in cysteine proteases might therefore result from a combination of functional and structural constraints.

Molecular Modeling and Site-directed Mutagenesis Define the Catalytic Motif in Human γ-Glutamyl Hydrolase

“…The thermal stability for the hGH E222A mutant protein was also decreased, and a lower yield of purified E222A was observed (results not shown). These observations are consistent with results obtained by site-directed mutagenesis of the putative third member of the catalytic triad of papain (36). Mutation of this asparagine to glutamine and alanine reduced the k cat /K m 3.4-and 150-fold, respectively.…”

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

“…Substrate-induced activation of sortase may be advantageous, preventing spurious proteolysis reactions without the need for more elaborate inactivation mechanisms, such as prosegment occlusion of the active site as observed in the zymogen structures of papain and cathepsin. In the cysteine proteases, a hydrogen bond between the side-chain oxygen of N175 and the N 2 of H159 has been proposed to stabilize the active site (49). Analogously, N98 in sortase may stabilize the positioning of H120, because in sortase the chemical shift positions of the imidazole nitrogens of H120 indicate that it is fully protonated, and 48% of the conformers in the ensemble contain a hydrogen bond between the N 2 proton of H120 to the side-chain oxygen of N98.…”

“…Analogously, N98 in sortase may stabilize the positioning of H120, because in sortase the chemical shift positions of the imidazole nitrogens of H120 indicate that it is fully protonated, and 48% of the conformers in the ensemble contain a hydrogen bond between the N 2 proton of H120 to the side-chain oxygen of N98. This interaction would not seem to be essential, because N98 is poorly conserved in the sortase family, and N175 in the cysteine proteases can be replaced by glutamine or alanine without complete loss of enzymatic activity (49).…”

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.…”

“…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.…”