Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains.
Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.
Lansdon
Eric B
EB
Department of Chemistry and Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, California 95616, USA.
Fisher
Andrew J
AJ
Segel
Irwin H
IH
D000250:Adenosine Phosphosulfate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001149:Arsenates / Q000737:chemistry; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D002857:Chromium / Q000737:chemistry; D006801:Humans; D043564:Inorganic Pyrophosphatase / Q000737:chemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D008982:Molybdenum / Q000737:chemistry; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D064586:Selenic Acid; D018036:Selenium Compounds / Q000737:chemistry; D013379:Substrate Specificity; D013430:Sulfate Adenylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D017973:Tungsten Compounds / Q000737:chemistry
D001149:Arsenates; D007527:Isoenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D018036:Selenium Compounds; D017973:Tungsten Compounds; D002857:Chromium; C044659:molybdate; C074702:chromium hexavalent ion; D000250:Adenosine Phosphosulfate; D008982:Molybdenum; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase; D043564:Inorganic Pyrophosphatase; D064586:Selenic Acid; C025657:arsenic acid; C045951:tungstate