The catalytic general base, Pro-1, of the enzyme 4-oxalocrotonate tautomerase has been mutated to Gly, Ala, Val, and Leu, residues with aliphatic side chains. The Val mutant was partially (55%) processed by removal of the amino-terminal methionine to yield P1V/M1P2V, while the Leu mutant was not processed and completely retained methionine (M1P2L). The M1P2L mutant lost 2300-fold in kcat with no change in Km, and the residual activity of the unresolvable P1V/M1P2V mixture could be explained by the summation of two activities, one equal to that of M1P2L and the other equal to that of the P1G mutant. The P1G and P1A mutants showed 76- and 58-fold decreases in kcat and much smaller decreases in Km 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 1H-15N HSQC spectra and 3D 1H-15N NOESY HSQC spectra of the 15N-labeled P1G mutant showed no structural differences from the wild-type enzyme except for small changes in backbone 15N and NH chemical shifts at the active site. Both the P1G and P1A mutants showed no change in overall conformation by circular dichroic spectroscopy. Both mutants and the wild-type enzyme generate the S-enantiomer of the product [5-2H]-2-oxo-3-hexenedioate with comparable stereoselectivities indicating a largely intact active site. The P1G and P1A mutants showed 10- and 4-fold decreases, respectively, 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 Ala-1 compared to Pro-1. The pH dependences of kcat/Km for the P1G and P1A mutants revealed pKa values of the general base of 5.3 and 5.9, respectively. NMR titration of the uniformly 15N-labeled P1G mutant showed the pKa of Gly-1 to be < or = 5.6, in agreement with the kinetic data. As with the wild-type enzyme, the active site environments on the P1G and P1A mutants lower the pKa of the general base by at least 2.5 units. It is concluded that the 2 order of magnitude decreases in kcat 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 kcat found with the presence of an additional residue at the amino-terminus is ascribed to either the complete blockage or the drastically altered position of the general base.