Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme

• Published in: Biochemistry (Easton) Vol. 26; no. 5; pp. 1258 - 1264
• Main Authors: Casal, Jose I, Ahern, Tim J, Davenport, Robert C, Petsko, Gregory A, Klibanov, Alexander M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 10.03.1987
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbohydrate Epimerases - genetics; Cloning, Molecular; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Immunologic Techniques; Isoelectric Point; Isomerases; Kinetics; Macromolecular Substances; Models, Molecular; Peptide Fragments; Protein Conformation; Protein Denaturation; Saccharomyces cerevisiae - enzymology; Structure-Activity Relationship; Triose-Phosphate Isomerase - genetics; Triose-Phosphate Isomerase - immunology; Triose-Phosphate Isomerase - metabolism; Yeast; Stability; Enzyme; Escherichia coli; Triosephosphate isomerase; Gene expression; Subunit; Fungi; Bacteria; Ascomycetes; Mutation; Escherichieae; Saccharomyces cerevisiae; Interface; Enterobacteriaceae; Thallophyta; Conformation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00379a009

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78---Asp-78 and the double replacement Asn-14/Asn-78---Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78---Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.