Kinetic, Stereochemical, and Structural Effects of Mutations of the Active Site Arginine Residues in 4-Oxalocrotonate Tautomerase

Published

July 1, 1999

Author(s)

T K. Harris, R M. Czerwinski, W H. Johnson, P M. Legler, C Abeygunawardana, M A. Massiah, J T. Stivers, C P. Whitman, A S. Mildvan

Abstract

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson Jr., R. M., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k_cat 88-fold and increased K_m 8.6-fold suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate 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 dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased kcat by 125- and 389-fold and increased K_m by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10⁴-fold lower than that of the wild type enzyme indicating approximate additivity of the effects of the two arginine mutants on kcat. For both R11A and R39Q, 2D ¹H- ¹⁵N HSQC and 3D ¹H-¹⁵N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the Β-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analog cis,cis-muconate yielded a K_d = 22 mmol/L, 37-fold greater than the Kd found with wild type 4-OT (0.6 mM). With the R39Q mutant, cis,cis-muconate showed negative 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 R39Q mutant. The ¹⁵Nξ resonances of all six Arg residues of 4-OT were assigned and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild type 4-OT upshifts Arg-11 Nξ (by 0.05 ppm) and downshifts Arg-39 Nξ (by 1.19 ppm), indicating 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 10⁷-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.

Citation

Biochemistry

Volume

38

Pub Type

Journals

Keywords

¹H-¹⁵N HSQC titrations, arginine, heteronucle NMR, negative cooperativity, Nξ assignments, proton transfer

Citation

Harris, T. , Czerwinski, R. , Johnson, W. , Legler, P. , Abeygunawardana, C. , Massiah, M. , Stivers, J. , Whitman, C. and Mildvan, A. (1999), Kinetic, Stereochemical, and Structural Effects of Mutations of the Active Site Arginine Residues in 4-Oxalocrotonate Tautomerase, Biochemistry (Accessed October 20, 2025)