Binding of 15 N-labeled isoniazid to KatG and KatG(S315T): Use of two- spin [zz]-order relaxation rate for 15 N-fe distance determination

Smilja Todorović, Nenad Juranić, Slobodan Macura, Frank Rusnak

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Isoniazid has been used to treat tuberculosis for over 40 years, but its mechanism of action is not yet fully understood. It is known that a catalase/peroxidase KatG is required for isoniazid activation. A point mutation in KatG, KatG(S315T), is found in > 50% of isoniazid-resistant strains. In this study we have measured the distance between the 15 N labeled amide nitrogen of isoniazid and the Fe ion in the active site of KatG and KatG(S315T). The distances are found to be equal within experimental error, 3.8 ± 0.8 and 4.4 ± 0.9 Å, respectively. A new method of measuring longitudinal relaxation rates of insensitive nuclei in paramagnetic systems via zz-order is proposed. The longitudinal relaxation rate of the 15 N nucleus was obtained from the independently measured longitudinal proton relaxation rate and the longitudinal zz-order relaxation rate of scalar coupled N and H atoms. To eliminate cross-correlations of different relaxation sources, a remote proton was used to create zz-order and detect the 15 N nucleus. The obtained 15 NFe distances are significantly shorter than previously reported 1 H-Fe distances (Wengenack, N. L.; Todorovic, S.; Yu, L; Rusnak, F. Biochemistry 1998, 37, 15825), indicating that the isoniazid molecule approaches the heme Fe ion via the hydrazine nitrogen atoms. The proposed method for two-spin order relaxation measurements is quite general and can be used to probe the distance between insensitive nuclei and a paramagnetic center in various protein-substrate complexes.

Original languageEnglish (US)
Pages (from-to)10962-10966
Number of pages5
JournalJournal of the American Chemical Society
Issue number47
StatePublished - Dec 1 1999

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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