Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme.
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.
D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics*; D003001:Cloning, Molecular; D007158:Immunologic Techniques; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D010446:Peptide Fragments; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D014305:Triose-Phosphate Isomerase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase