Journal of Protein Chemistry

, 17:579

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

  • Kazumi Funane
  • Nathalie Libessart
  • Douglas Stewart
  • Toru Michishita
  • Jack Preiss
Article

DOI: 10.1007/BF02780959

Cite this article as:
Funane, K., Libessart, N., Stewart, D. et al. J Protein Chem (1998) 17: 579. doi:10.1007/BF02780959

Abstract

Incubation of maize branching enzyme, mBEI and mBEII, with 100 µM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadKm values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.

Key words

Branching enzymechemical modificationdiethyl pyrocarbonatesite-directed mutagenesishistidine residuessubstrate binding

Copyright information

© Plenum Publishing Corporation 1998

Authors and Affiliations

  • Kazumi Funane
    • 1
  • Nathalie Libessart
    • 1
  • Douglas Stewart
    • 1
  • Toru Michishita
    • 1
  • Jack Preiss
    • 1
  1. 1.Department of BiochemistryMichigan State UniversityEast Lansing
  2. 2.National Food Research InstituteIbarakiJapan
  3. 3.Department of Plant Science, Waite Agricultural Research InstituteUniversity of AdelaideGlen OsmondAustralia
  4. 4.Agricultural Research InstituteHokuren Federation of Agricultural Cooperatives E7, N6Higashi-ku, SapporoJapan