Direct evidence for the exploitation of an alpha-helix in the catalytic mechanism of triosephosphate isomerase.

In previous work, we have shown that the first (and, presumably, the second) pKa of the active-site histidine-95 in triosephosphate isomerase has been lowered by about 2 units [Lodi, P. J., & Knowles, J. R. (1991) Biochemistry 30, 6948-6956]. One reason for the perturbed pKa of this residue appears to be its location at the N-terminus of a short alpha-helix that runs from residues 95 to 102. Fortuitously, the residue at the C-terminus of this helix is also a histidine residue (histidine-103), and the existence of a histidine side chain at each end has allowed us directly to implicate the helix in the perturbation of the pKa value of histidine-95. 15N NMR titration studies of the native enzyme and 13C NMR titration studies of the denatured enzyme show that while the pKa of histidine-95 is lowered by a least 2 units in the folded versus the unfolded state, the pKa of histidine-103 is raised by about 0.6 unit on protein folding. These complementary effects on the pKa values of histidine-95 and histidine-103 suggest that the alpha-helix is indeed responsible for the perturbation of the pKa values. The larger effect on the pKa of histidine-95 is readily rationalized in terms of the local structure of the enzyme. The disparity in the perturbation for the two histidine side chains illustrates how an alpha-helix can be functionally utilized by proteins, directly to affect (as in the present case) the chemistry of catalysis by an enzyme.