Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain

The existence of an oxyanion hole in cysteine proteases able to stabilize a transition-state complex in a manner analogous to that found with serine proteases has been the object of controversy for many years. In papain, the side chain of Gln19 forms one of the hydrogen-bond donors in the putative oxyanion hole, and its contribution to transition-state stabilization has been evaluated by site-directed mutagenesis. Mutation of Gln19 to Ala caused a decrease in k(cat)/K(M) for hydrolysis of CBZ-Phe-Arg-MCA, which is 7700 M-1 s-1 in the mutant enzyme as compared to 464 000 M-1 s-1 in wild-type papain. With a Gln19ser variant, the activity is even lower, with a k(cat)/K(M) value of 760 M-1 s-1. The 60- and 600-fold decreases in k(cat)/K(M) correspond to changes in free energy of catalysis of 2.4 and 3.8 kcal/mol for Gln19Ala and Gln19Ser, respectively. In both cases, the decrease in activity is in large part attributable to a decrease in k(cat), while K(M) values are only slightly affected. These results indicate that the oxyanion hole is operational in the papain-catalyzed hydrolysis of CBZ-Phe-Arg-MCA and constitute the first direct evidence of a mechanistic requirement for oxyanion stabilization in the transition state of reactions catalyzed by cysteine proteases. The equilibrium constants Ki for inhibition of the papain mutants by the aldehyde Ac-Phe-Gly-CHO have also been determined. Contrary to the results with the substrate, mutation at position 19 of papain has a very small effect on binding of the inhibitor. This important finding indicates that different types of inhibitors and substrates utilize the catalytic machinery of papain to differing degrees. Implications for the role of oxyanion hole interactions in cysteine proteases are discussed