Triosephosphate Isomerase Requires a Positively Charged Active Site: The Role of Lysine-12

• Published in: Biochemistry (Easton) Vol. 33; no. 10; pp. 2809 - 2814
• Main Authors: Lodi, Patricia J, Chang, Louise C, Knowles, Jeremy R, Komives, Elizabeth A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-03-1994
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Crystallography, X-Ray; DNA Primers; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; ISOMERASAS; ISOMERASE; Isomerases; Kinetics; LISINA; LYSINE; Methionine; Molecular Sequence Data; MUTACION; MUTACION INDUCIDA; Mutagenesis, Site-Directed; MUTANT; MUTANTES; MUTATION; MUTATION PROVOQUEE; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Spectroscopy, Fourier Transform Infrared; Triose-Phosphate Isomerase - chemistry; Triose-Phosphate Isomerase - metabolism; Ligand binding; Yeast; Intramolecular oxidoreductases; Enzyme; Escherichia coli; Active site; Triose-phosphate isomerase; Isomerases; Enzymatic activity; Structure activity relation; Bacteria; Kinetic parameter; Recombinant protein; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00176a009

The role of lysine-12 at the active site of yeast triosephosphate isomerase has been elucidated by a combination of site-directed mutagenesis, Fourier transform infrared spectroscopy, enzyme kinetics, and X-ray crystallography. Several lines of evidence suggest that the mutant isomerase in which lysine has been changed to methionine cannot bind substrate. This mutant enzyme has no detectable catalytic activity, and infrared experiments show no evidence of binding dihydroxyacetone phosphate nor dihydroxyacetone sulfate to the active site. Furthermore, crystals of the enzyme grown in the presence of phosphoglycolohydroxamate, a potent reaction intermediate analog, show an open active site with no inhibitor bound. Mutation of lysine-12 to arginine produces a protein with a value Km elevated by a factor of 22, a Vmax reduced by a factor of 180, and a Ki for phosphoglycolohydroxamate elevated by a factor of 290. Mutation of lysine-12 to histidine produces an enzyme that shows virtually no catalytic activity at neutral pH, but below pH 6.1 this enzyme is active, suggesting that protonation of the histidine in this mutant is required for activity. These studies, together with the structural results reported in an accompanying paper, provide convincing evidence that a positive charge is required for substrate binding at the active site of triosephosphate isomerase and that lysine-12 provides this positive charge