Mass spectrometric analysis of phosphate from β,γ-[18O]ATP hydrolyzed by Azotobacter vinelandii nitrogenase

Direct evidence for PγOPβ bond cleavage

Charles E. McKenna, William G. Gutheil, George L. Kenyon, Terry O Matsunaga

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Nitrogenase catalyzes reduction-dependent hydrolysis of ATP to ADP and phosphate. It has been generally assumed that the catalytic process involves attack of a nucleophile at the Pγ of ATP by analogy with conventional ATPases, but no direct evidence verifying this assumption has been available. To determine which anhydride PO bond is cleaved by the enzyme, β,γ-[18O]ATP (75.4% specific enrichment; nonspecific enrichments of 6.0% nonbridging Pγ18O, 4.0% nonbridging Pβ18O, and 2.0% Pβ18OPα) was incubated with purified nitrogenase proteins from Azotobacter vinelandii under turnover conditions. The phosphate produced was isolated, derivatized to trimethyl phosphate, and analyzed by high-resolution mass spectrometry. The C3H9O4P C3H9O318OP mass ( 140 142) ratio found in the product phosphate derivative was 14.0 ± 2.0 (SE, n = 3). The calculated values for PγOPβ and PγOPβ bond breaking based on the known distribution of 18O in the substrate were 15 and 0.23, respectively. The results establish nitrogenase hydrolysis of the ATP PγOPβ linkage.

Original languageEnglish (US)
Pages (from-to)377-384
Number of pages8
JournalBioorganic Chemistry
Volume17
Issue number4
DOIs
StatePublished - 1989
Externally publishedYes

Fingerprint

Azotobacter vinelandii
Nitrogenase
Adenosine Triphosphate
Phosphates
Hydrolysis
Nucleophiles
Anhydrides
Adenosine Diphosphate
Mass spectrometry
Adenosine Triphosphatases
Mass Spectrometry
Derivatives
Substrates
Enzymes
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Organic Chemistry
  • Drug Discovery

Cite this

Mass spectrometric analysis of phosphate from β,γ-[18O]ATP hydrolyzed by Azotobacter vinelandii nitrogenase : Direct evidence for PγOPβ bond cleavage. / McKenna, Charles E.; Gutheil, William G.; Kenyon, George L.; Matsunaga, Terry O.

In: Bioorganic Chemistry, Vol. 17, No. 4, 1989, p. 377-384.

Research output: Contribution to journalArticle

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abstract = "Nitrogenase catalyzes reduction-dependent hydrolysis of ATP to ADP and phosphate. It has been generally assumed that the catalytic process involves attack of a nucleophile at the Pγ of ATP by analogy with conventional ATPases, but no direct evidence verifying this assumption has been available. To determine which anhydride PO bond is cleaved by the enzyme, β,γ-[18O]ATP (75.4{\%} specific enrichment; nonspecific enrichments of 6.0{\%} nonbridging Pγ18O, 4.0{\%} nonbridging Pβ18O, and 2.0{\%} Pβ18OPα) was incubated with purified nitrogenase proteins from Azotobacter vinelandii under turnover conditions. The phosphate produced was isolated, derivatized to trimethyl phosphate, and analyzed by high-resolution mass spectrometry. The C3H9O4P C3H9O318OP mass ( 140 142) ratio found in the product phosphate derivative was 14.0 ± 2.0 (SE, n = 3). The calculated values for PγOPβ and PγOPβ bond breaking based on the known distribution of 18O in the substrate were 15 and 0.23, respectively. The results establish nitrogenase hydrolysis of the ATP PγOPβ linkage.",
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