TY - JOUR
T1 - Mass spectrometric analysis of phosphate from β,γ-[18O]ATP hydrolyzed by Azotobacter vinelandii nitrogenase
T2 - Direct evidence for PγOPβ bond cleavage
AU - McKenna, Charles E.
AU - Gutheil, William G.
AU - Kenyon, George L.
AU - Matsunaga, Terry O.
N1 - Funding Information:
r Supported by U. S. Public Health Service Grants GM-31716 from the National Institute of General Medicine (C.E.M.), AR-17323 from the National Institute of Arthritis, Metabolism and Digestive Diseases (G.L.K.); Grant 84-CRCR-l-1106 from USDA-CRGO (C.E.M.); and Grant HF-67 from the H. Frasch Foundation (C.E.M.). A preliminary report of this work has appeared in abstract form (1). * To whom correspondence should be addressed. 3 Present address: Department of Chemistry, University of Arizona, Tuscan, AZ 85721. 377
PY - 1989/12
Y1 - 1989/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0024806424&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0024806424&partnerID=8YFLogxK
U2 - 10.1016/0045-2068(89)90039-4
DO - 10.1016/0045-2068(89)90039-4
M3 - Article
AN - SCOPUS:0024806424
SN - 0045-2068
VL - 17
SP - 377
EP - 384
JO - Bioorganic Chemistry
JF - Bioorganic Chemistry
IS - 4
ER -