ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm).
ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.
Renosto
F
F
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Martin
R L
RL
Borrell
J L
JL
Nelson
D C
DC
Segel
I H
IH
D000250:Adenosine Phosphosulfate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D001665:Binding Sites; D002412:Cations; D003545:Cysteine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D008982:Molybdenum / Q000378:metabolism; D009994:Osmolar Concentration; D011077:Polychaeta / Q000201:enzymology*; D011487:Protein Conformation; D013430:Sulfate Adenylyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature
D002412:Cations; C044659:molybdate; D000250:Adenosine Phosphosulfate; D008982:Molybdenum; D000255:Adenosine Triphosphate; D013430:Sulfate Adenylyltransferase; D003545:Cysteine