The structural basis for the perturbed pK(a) of the catalytic base in 4-oxalocrotonate tautomerase: Kinetic and structural effects of mutations of Phe-50

The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of beta ,gamma -unsaturated enones to their alpha,beta -isomers because of its unus ually low pK(a) of 6.4 +/- 0.2, which is 3 units lower than that of the mod el compound, proline amide. Recent studies show that this abnormally low pK (a) is not due to the electrostatic effects of nearby cationic residues (Ar g-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1 999) Biochemistry 38, 12358-1.2366]. Hence, it may result solely from a low local dielectric constant of 14.7 +/- 0.8 at the otherwise hydrophobic act ive site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 Angstrom from Pro-1 and is one of 12 apol ar residues within 9 Angstrom of Pro-1. Replacing Phe-50 with Tyr does not significantly alter k(cat) or K-m and results in a pK(a) of 6.0 +/- 0.1 for Pro-1 as determined by N-15 NMR spectroscopy, comparable to that observed for wild type. H-1-N-15 HSQC and 3D H-1-N-15 NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild -type enzyme. In the F50Y mutant, the pK(a) of Tyr-50 is increased by two u nits from that of a model compound N-acetyl-tyrosine amide to 12.2 +/- 0.3, as determined by W and H-1 NMR titrations, yielding a local dielectric con stant of 13.4 +/- 1.7, in agreement with the value of 13.7 +/- 0.3 determin ed from the decreased pK(a) of Pro-i in this mutant. In the F50A mutant, th e pK(a) of Pro-1 is 7.3 +/- 0.1 by N-15 NMR titration, comparable to the pK (a) of 7.6 +/- 0.2 found in the pH vs k(cat)/K-m rate profile, and is one u nit greater than that of the wild-type enzyme, indicating an increase in th e local dielectric constant to a value of 21.2 +/- 2.6. A loss of structure of the beta -hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4- OT. H-1-N-15 HSQC spectra of the F50A mutant reveal widespread and large ch anges in the backbone N-15 and NH chemical shifts including those of Gly re sidues 48, 51, 53, and 54 causing their loss of dispersion at 23 degreesC a nd their disappearance at 43 degreesC due to rapid exchange with solvent. T hese observations confirm that the active site of the F50A mutant is more a ccessible to the external aqueous environment, causing an increase in the l ocal dielectric constant and in the pK(a) of Pro-1. In addition, the F50A m utation decreased k(cat) 167-fold and increased K-m 11-fold from those of t he wildtype enzyme, suggesting an important role for the hydrophobic enviro nment in catalysis, beyond that of decreasing the pK(a) of Pro-1. The F50I and F50V mutations destabilize the protein and decrease k(cat) by factors o f 58 and 1.6, and increase K-m by 3.3- and 3.8-fold, respectively.