title: Direct evidence for the exploitation of an alpha-helix in the catalytic mechanism of triosephosphate isomerase.
abstract: 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.
authors: Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
authors: P J
authors: PJ
authors: Lodi
authors: J R
authors: JR
authors: Knowles
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D014305:Triose-Phosphate Isomerase