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 kcat/KM 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 kcat/KM value of 760 M-1 s-1. The 60- and 600-fold decreases in kcat/KM 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 kcat, while KM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ménard
R
R
Biotechnology Reserach Institute, National Research Council of Canada, Montréal, Québec.
Carrière
J
J
Laflamme
P
P
Plouffe
C
C
Khouri
H E
HE
Vernet
T
T
Tessier
D C
DC
Thomas
D Y
DY
Storer
A C
AC
D000595:Amino Acid Sequence; D000838:Anions; D001483:Base Sequence; D002384:Catalysis; D005973:Glutamine / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010206:Papain / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity
D000838:Anions; D005973:Glutamine; D010206:Papain