Rat liver ATP-sulfurylase: purification, kinetic characterization, and interaction with arsenate, selenate, phosphate, and other inorganic oxyanions.
ATP-sulfurylase (ATP:sulfate adenylyltransferase; EC 2.7.7.4), the first enzyme of the two-step sulfate activation sequence, was purified extensively from rat liver cytosol. The enzyme has a native molecular mass of 122 +/- 12 kDa and appears to be composed of identical 62 +/- 6-kDa subunits. At 30 degrees C and pH 8.0 (50 mM Tris-Cl buffer containing 5 mM excess Mg2+), the best preparations have "forward reaction" specific activities of about 20 and 2 units X mg protein-1 with MoO4(2-) and SO4(2-), respectively. The reverse (ATP synthesis) specific activity is about the same as the forward molybdolysis activity. The kinetic constants under the above conditions are as follows: KmA = 0.21 mM, Kia = 0.87 mM, KmB = 0.18 mM, KmQ = 0.65 microM, Kiq = 0.11 microM, and KmP = 5.0 microM where A = MgATP, B = SO4(2-), Q = APS, and P = total PPi at 5 mM Mg2+. PPi is a mixed-type inhibitor with respect to MgATP and SO4(2-). SeO4(2-) is an alternative inorganic substrate with a Vmax about 20% that of SO4(2-). The product, APSe, is unstable. But in the presence of a sufficient excess of APS kinase, APSe is completely converted to PAPSe. The rate constant for nonenzymatic PAPSe hydrolysis was determined from measurements of the final steady-state reaction rate in the presence of limiting initial SeO4(2-) and a large excess of MgATP, ATP sulfurylase, APS kinase, and the other coupling enzymes and their cosubstrates. The results yielded a k of 2.4 +/- 0.5 X 10(-3) sec-1 (t1/2 ca. 5 min). Phosphate is an effective buffer for enzyme purification and storage but inhibits catalytic activity, particularly at low substrate concentrations. In the presence of buffer levels of Pi, the MgATP reciprocal plot of the SO4(2-)-dependent reaction is concave-up. Inorganic monovalent oxyanions are dead end inhibitors competitive with SO4(2-) and apparently uncompetitive with respect to MgATP. The relative potencies are in the order ClO3- greater than ClO4- greater than FSO3- greater than NO3-. Thiosulfate is also competitive with SO4(2-) but noncompetitive with respect to MgATP. Several divalent oxyanions (MoO4(2-), WO4(2-), CrO4(2-), and HAsO4(2-] promote the enzyme-catalyzed cleavage of MgATP to AMP and MgPPi. The ratio Vmaxf/KmA ranged from 0.7 to 200 for various reactive inorganic substrates. The cumulative results suggest the random binding of MgATP and the inorganic substrate but the ordered release of MgPPi before APS.(ABSTRACT TRUNCATED AT 400 WORDS)
Yu
M
M
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Martin
R L
RL
Jain
S
S
Chen
L J
LJ
Segel
I H
IH
D000250:Adenosine Phosphosulfate / Q000096:biosynthesis; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D000838:Anions / Q000378:metabolism* / Q000494:pharmacology; D001149:Arsenates / Q000378:metabolism / Q000494:pharmacology; D002413:Cations, Divalent; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D011919:Rats, Inbred Strains; D064586:Selenic Acid; D012643:Selenium / Q000378:metabolism / Q000494:pharmacology; D018036:Selenium Compounds; D013379:Substrate Specificity; D013430:Sulfate Adenylyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature
D000838:Anions; D001149:Arsenates; D002413:Cations, Divalent; D010710:Phosphates; D018036:Selenium Compounds; D000250:Adenosine Phosphosulfate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D009713:Nucleotidyltransferases; D013430:Sulfate Adenylyltransferase; D012643:Selenium; D064586:Selenic Acid; C025657:arsenic acid