Human 3′-Phosphoadenosine 5′-Phosphosulfate Synthetase (Isoform 1, Brain): Kinetic Properties of the Adenosine Triphosphate Sulfurylase and Adenosine 5′-Phosphosulfate Kinase Domains

Eric B. Lansdon, Andrew J Fisher, Irwin H. Segel

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Abstract

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 (K1 of 47.9 μM at saturating MgATP). The maximum attainable activity of 0.12 μmol min-1 (mg of protein)-1 was observed at an APS concentration ([APS] opt) of 15 μM. The theoretical Km for APS (at saturating MgATP) and the Km for MgATP (at [APS] opt) were 4.2 μM 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 μmol min-1 (mg of protein)-1. Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 μmol min-1 (mg of protein)-1. The kinetic constants of the ATP sulfurylase domain were as follows: Vmax,f = 0.77 μmol min-1 (mg of protein)-1, KmA(MgATP) = 0.15 mM, Kia(MgATP) = 1 mM, KmB(sulfate) = 0.16 mM, Vmax,r = 18.7 μmol min-1 (mg of protein)-1, KmQ(APS) = 4.8 μM, Kiq(APS) = 18 nM, and KmP(PPi) = 34.6 μM. 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) availablity 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 PPi. 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.

Original languageEnglish (US)
Pages (from-to)4356-4365
Number of pages10
JournalBiochemistry
Volume43
Issue number14
DOIs
StatePublished - Apr 13 2004

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adenylylsulfate kinase
Sulfate Adenylyltransferase
Adenosine Phosphosulfate
Ligases
Brain
Protein Isoforms
Adenosine Triphosphate
Kinetics
Phosphoadenosine Phosphosulfate
Selenic Acid
Chlorates
Sulfates
Chromates
Proteins
PAPS synthetase
Penicillium chrysogenum

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{be0157f3bdae47e38c3c0d7a7b333806,
title = "Human 3′-Phosphoadenosine 5′-Phosphosulfate Synthetase (Isoform 1, Brain): Kinetic Properties of the Adenosine Triphosphate Sulfurylase and Adenosine 5′-Phosphosulfate Kinase Domains",
abstract = "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 (K1 of 47.9 μM at saturating MgATP). The maximum attainable activity of 0.12 μmol min-1 (mg of protein)-1 was observed at an APS concentration ([APS] opt) of 15 μM. The theoretical Km for APS (at saturating MgATP) and the Km for MgATP (at [APS] opt) were 4.2 μM 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 μmol min-1 (mg of protein)-1. Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 μmol min-1 (mg of protein)-1. The kinetic constants of the ATP sulfurylase domain were as follows: Vmax,f = 0.77 μmol min-1 (mg of protein)-1, KmA(MgATP) = 0.15 mM, Kia(MgATP) = 1 mM, KmB(sulfate) = 0.16 mM, Vmax,r = 18.7 μmol min-1 (mg of protein)-1, KmQ(APS) = 4.8 μM, Kiq(APS) = 18 nM, and KmP(PPi) = 34.6 μM. 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) availablity 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 PPi. 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.",
author = "Lansdon, {Eric B.} and Fisher, {Andrew J} and Segel, {Irwin H.}",
year = "2004",
month = "4",
day = "13",
doi = "10.1021/bi049827m",
language = "English (US)",
volume = "43",
pages = "4356--4365",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "14",

}

TY - JOUR

T1 - Human 3′-Phosphoadenosine 5′-Phosphosulfate Synthetase (Isoform 1, Brain)

T2 - Kinetic Properties of the Adenosine Triphosphate Sulfurylase and Adenosine 5′-Phosphosulfate Kinase Domains

AU - Lansdon, Eric B.

AU - Fisher, Andrew J

AU - Segel, Irwin H.

PY - 2004/4/13

Y1 - 2004/4/13

N2 - 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 (K1 of 47.9 μM at saturating MgATP). The maximum attainable activity of 0.12 μmol min-1 (mg of protein)-1 was observed at an APS concentration ([APS] opt) of 15 μM. The theoretical Km for APS (at saturating MgATP) and the Km for MgATP (at [APS] opt) were 4.2 μM 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 μmol min-1 (mg of protein)-1. Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 μmol min-1 (mg of protein)-1. The kinetic constants of the ATP sulfurylase domain were as follows: Vmax,f = 0.77 μmol min-1 (mg of protein)-1, KmA(MgATP) = 0.15 mM, Kia(MgATP) = 1 mM, KmB(sulfate) = 0.16 mM, Vmax,r = 18.7 μmol min-1 (mg of protein)-1, KmQ(APS) = 4.8 μM, Kiq(APS) = 18 nM, and KmP(PPi) = 34.6 μM. 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) availablity 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 PPi. 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.

AB - 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 (K1 of 47.9 μM at saturating MgATP). The maximum attainable activity of 0.12 μmol min-1 (mg of protein)-1 was observed at an APS concentration ([APS] opt) of 15 μM. The theoretical Km for APS (at saturating MgATP) and the Km for MgATP (at [APS] opt) were 4.2 μM 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 μmol min-1 (mg of protein)-1. Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 μmol min-1 (mg of protein)-1. The kinetic constants of the ATP sulfurylase domain were as follows: Vmax,f = 0.77 μmol min-1 (mg of protein)-1, KmA(MgATP) = 0.15 mM, Kia(MgATP) = 1 mM, KmB(sulfate) = 0.16 mM, Vmax,r = 18.7 μmol min-1 (mg of protein)-1, KmQ(APS) = 4.8 μM, Kiq(APS) = 18 nM, and KmP(PPi) = 34.6 μM. 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) availablity 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 PPi. 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.

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