Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T)

Biochemistry. 1998 Nov 10;37(45):15825-34. doi: 10.1021/bi982023k.


Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.

MeSH terms

  • Amino Acid Substitution / genetics
  • Bacterial Proteins*
  • Drug Resistance, Microbial / genetics
  • Electron Spin Resonance Spectroscopy
  • Isoniazid / chemistry
  • Isoniazid / metabolism*
  • Isoniazid / pharmacology
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Mutagenesis, Site-Directed
  • Mycobacterium tuberculosis / drug effects
  • Mycobacterium tuberculosis / metabolism*
  • Peroxidases / chemistry
  • Peroxidases / genetics*
  • Peroxidases / metabolism*
  • Protein Binding
  • Serine / genetics
  • Spectrophotometry
  • Threonine / genetics


  • Bacterial Proteins
  • Threonine
  • Serine
  • Peroxidases
  • catalase HPI
  • Isoniazid