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

• Published in: Biochemistry (Easton) Vol. 32; no. 16; pp. 4338 - 4343
• Main Authors: Lodi, Patricia J, Knowles, Jeremy R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 27-04-1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Cloning, Molecular; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetic Vectors; Histidine; Hydrogen-Ion Concentration; Isomerases; Kinetics; Magnetic Resonance Spectroscopy - methods; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Protein Folding; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Thermodynamics; Triose-Phosphate Isomerase - chemistry; Triose-Phosphate Isomerase - genetics; Triose-Phosphate Isomerase - metabolism; Enzymatic activity; Enzyme; Active site; Triosephosphate isomerase; Reaction mechanism; Helical structure; Catalysis
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00067a024

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.