Current Microbiology

, Volume 35 , Issue 1 , pp 14 –17

Lysine 194 Is Functional in Isocitrate Lyase from Escherichia coli

Authors

  • Abdur  Rehman
    • Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660, USA
  • Bruce A.  McFadden
    • Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660, USA

DOI: 10.1007/s002849900203

Cite this article as:
Rehman, A. & McFadden, B. Curr Microbiol (1997) 35: 14. doi:10.1007/s002849900203

Abstract.

Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had kcat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. Km values for Mg2+-Ds-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased Km value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state.

Copyright information

© Springer-Verlag New York Inc. 1997