Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme. Academic Article uri icon

Overview

abstract

  • 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.

publication date

  • March 10, 1987

Research

keywords

  • Carbohydrate Epimerases
  • Triose-Phosphate Isomerase

Identity

Scopus Document Identifier

  • 0023134941

Digital Object Identifier (DOI)

  • 10.1021/bi00379a009

PubMed ID

  • 3552044

Additional Document Info

volume

  • 26

issue

  • 5