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

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

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
Volume121
Issue number47
DOIs
StatePublished - Dec 1 1999

ASJC Scopus subject areas

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

Fingerprint Dive into the research topics of 'Binding of <sup>15</sup> N-labeled isoniazid to KatG and KatG(S315T): Use of two- spin [zz]-order relaxation rate for <sup>15</sup> N-fe distance determination'. Together they form a unique fingerprint.

  • Cite this