KINETIC AND STRUCTURAL EFFECTS OF MUTATIONS OF THE CATALYTIC AMINO-TERMINAL PROLINE IN 4-OXALOCROTONATE TAUTOMERASE

The catalytic general base, Pro-1, of the enzyme 4-oxalocrotonate taut omerase has been mutated to Gly, Ala, Val, and Leu, residues with alip hatic side chains. The Val mutant was partially (55%) processed by rem oval of the amino-terminal methionine to yield P1V/M1P2V, while the Le u mutant was not processed and completely retained methionine (M1P2L). The M1P2L mutant lost 2300-fold in k(cat) with no change in K-m, and the residual activity of the unresolvable P1V/M1P2V mixture could be e xplained by the summation of two activities, one equal to that of M1P2 L and the other equal to that of the P1G mutant. The P1G and P1A mutan ts showed 76- and 58-fold decreases in k(cat) and much smaller decreas es in K-m of 4- and 2.8-fold, respectively. The dissociation constant of the substrate analog cis,cis-muconate decreased 1.7-fold in the P1G mutant as determined by NMR titration. 2D H-1-N-15 HSQC spectra and 3 D H-1-N-15 NOESY HSQC spectra of the N-15-labeled P1G mutant showed no structural differences from the wildtype enzyme except for small chan ges in backbone N-15 and NH chemical shifts at the active site. Both t he P1G and P1A mutants showed no change in overall conformation by cir cular dichroic spectroscopy. Both mutants and the wild-type enzyme gen erate the S-enantiomer of the product [5-H-2]-2-oxo-3-hexenedioate wit h comparable stereoselectivities indicating a largely intact active si te. The P1G and P1A mutants showed 10- and 4-fold decreases, respectiv ely, in catalysis of exchange of the C3 proton of the substrate 2-oxo- 1,6-hexanedioate, consistent with the lower basicities of Gly-1 and Al a-1 compared to Pro-1. The pH dependences of k(cat)/K-m for the P1G an d P1A mutants revealed pK(a) values of the general base of 5.3 and 5.9 , respectively. NMR titration of the uniformly N-15-labeled P1G mutant showed the pK(a) of Gly-1 to be less than or equal to 5.6, in agreeme nt with the kinetic data. As with the wild-type enzyme, the active sit e environments on the P1G and P1A mutants lower the pK(a) of the gener al base by at least 2.5 units. It is concluded that the 2 order of mag nitude decreases in k(cat) in the P1G and P1A mutants result from both a decrease in basicity and an increase in flexibility of the general base. The greater 10(3.4)-fold decrease in k(cat) found with the prese nce of an additional residue at the amino-terminus is ascribed to eith er the complete blockage or the drastically altered position of the ge neral base.