Kinetic, stereochemical, and structural effects of mutations of the activesite arginine residues in 4-oxalocrotonate tautomerase

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active si te of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-l abeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitm an, C. P., and Hackert, M, L, (1998) Biochemistry 37, 14692-14700]. The cat alytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuc onate), the R61A mutation showed no kinetic effects, while the R11A mutatio n decreased k(cat) 88-fold and increased K-m 8.6-fold, suggesting both bind ing and catalytic roles for Arg-11, With a 1-monocarboxylate substrate (2-h ydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were fou nd, indicating that Arg-11 interacts with the 6-carboxylate of the substrat e, The stereoselectivity of the R11A-catalyzed protonation at C-5 of the di carboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild- type 4-OT, indicating the importance of Arg-11 in properly orienting the di carboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125 - and 389-fold and increased K-m by 1.5- and 2.6-fold, respectively, sugges ting a largely catalytic role for Arg-39. The activity of the R11A/R39A dou ble mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutan ts on k(cat), For both R11A and R39Q,2D H-1-N-15 HSQC and 3D H-1-N-15 NOESY -HSQC spectra showed chemical shift changes mainly near the mutated residue s, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis,cis -muconate yielded a K-d of 22 mM, 37-fold greater than the K-d found with w ild-type 4-OT (0.6 mM), With the R39Q mutant, cis,cis-muconate showed negat ive cooperativity in active site binding with two K-d values, 3.5 and 29 mM , This observation together with the low K-m of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R 39Q mutant. The N-15 epsilon resonances of all six Arg residues of 4-OT wer e assigned, and the assignments of Arg-11, -39, and -61 were confirmed by m utagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg- 11 N epsilon (by 0.05 ppm) and downshifts Arg-39 N epsilon (by 1.19 ppm), i ndicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10 (7)-fold catalytic power of 4-OT. Despite its presence in the active site i n the crystal structure of the affinity-labeled enzyme, Arg-61 does not pla y a significant role in either substrate binding or catalysis.