Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

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



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


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 kcat 88-fold and increased Km 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 Km 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 Kd = 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 Kd values, 3.5 and 29 MM. This observation together with the low Km 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 kcat, 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.


<sup>1</sup>H-<sup>15</sup>N HSQC titrations, arginine, heteronucle NMR, negative cooperativity, N&#958; assignments, proton transfer


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 June 22, 2024)


If you have any questions about this publication or are having problems accessing it, please contact

Created June 30, 1999, Updated October 12, 2021