Ser-143 is an essential active site residue in histidine ammonia-lyase of Pseudomonas putida

Biochem Biophys Res Commun. 1994 Jun 30;201(3):1433-8. doi: 10.1006/bbrc.1994.1863.

Abstract

Site directed mutagenesis was used to investigate the role of Ser-143 in enzyme activity and as a point for attack by cyanide or L-cysteine, two irreversible inhibitors of histidine ammonia-lyase (histidase). Two mutant proteins, a S143A substitution and an A142S-S143A transposition, were made. Both mutant histidases completely lost all enzymatic activity. Western blots with anti-histidase antibodies revealed that the mutant proteins were being expressed at a level equal to that of the wild-type protein. The purified mutant proteins could not incorporate [14C]cyanide nor could they generate the UV-absorbing species normally observed when L-cysteine modifies wild-type histidase. These results support the hypothesis that Ser-143 is the binding site for an as yet unidentified histidase cofactor.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Catalysis
  • Histidine Ammonia-Lyase / chemistry*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Pseudomonas putida / enzymology*
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Spectrophotometry, Ultraviolet
  • Structure-Activity Relationship

Substances

  • Histidine Ammonia-Lyase