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

Smilja Todorović, Nenad Juranić, Slobodan I Macura, Frank Rusnak

Research output: Contribution to journalArticle

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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 15N 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 15N 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 15N nucleus. The obtained 15NFe distances are significantly shorter than previously reported 1H-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

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Isoniazid
Protons
Nitrogen
Atoms
Biochemistry
Hydrazine
Ions
Amides
hydrazine
Chemical activation
Proteins
Molecules
Substrates
Heme
Point Mutation
Catalase
Peroxidase
Catalytic Domain
Tuberculosis

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Binding of 15N-labeled isoniazid to KatG and KatG(S315T) : Use of two- spin [zz]-order relaxation rate for 15N-fe distance determination. / Todorović, Smilja; Juranić, Nenad; Macura, Slobodan I; Rusnak, Frank.

In: Journal of the American Chemical Society, Vol. 121, No. 47, 01.12.1999, p. 10962-10966.

Research output: Contribution to journalArticle

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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 15N 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 {\AA}, 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 15N 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 15N nucleus. The obtained 15NFe distances are significantly shorter than previously reported 1H-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.",
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