Progress 10/01/02 to 09/30/07
Outputs
Inorganic sulfate from soil and water serves as the sole sulfur source for most plants and aerobic microorganisms. Plants and microbes use sulfate to produce cysteine and methionine and for the formation of several coenzymes such as CoA, biotin and thiamin. Animals use sulfate to form sulfate esters such as chondroitin sulfate, heparin, and sulfated hormones. Sulfate activation proceeds in two steps catalyzed by the sequential action of the enzymes ATP sulfurylase and APS kinase. The reactions produce, in order, the sulfonucleotides APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). In many chemo- and photolithotrophic bacteria, ATP sulfurylase catalyzes the last reaction in the oxidation of reduced inorganic sulfur compounds to sulfate i.e., the physiological reaction is in the opposite direction compared to that in sulfate assimilators. Our results to date indicate that ATP sulfurylases from sulfur chemolithotrophs are kinetically
optimized to release sulfate into a high sulfate environment. e.g., the enzymes have very poor affinities for sulfate. The enzymes from sulfate assimilators appear to be kinetically optimized to work best in the direction of ATP and sulfate formation. To date, the structures and kinetics of the enzyme from several chemolithotrophs have been established. These sources include the Riftia pachyptila (hydrothermal vent tube worm) symbiot and Aquifex aeolicus. So far, we have been unable to identify structural differences underlying the kinetic optimization. However, both of the chemolithotroph enzymes that have been examined are marine organisms and Aquifex is a thermophile. In order to complete the study, the structure of the enzyme from Thiobacillus denitrificans is being determined. This last organism is a terrestrial mesophilic chemolithotroph and as such, the structure of its ATP sulfurylase will control for interactions that are related to the marine environment or thermal
stability. Other research focused on the kinetic effects of mutating Asp-434 of the allosteric P. chrysogenum enzyme.. Asp-434 was chosen because it is located within the allosteric site cavity and is completely conserved in all fungal ATP sulfurylases. But Asp434 has no obvious function in binding the allosteric inhibitor, PAPS. In the absence of PAPS, Asp434Ala resembled the wild type enzyme kinetically. But the Asp434Ala mutant was hypersensitive to PAPS. For example, under standard assay condition where the wild type shows an IC50 of ca. 30 uM, Asp434Ala had an IC50 of 150 nM. This result suggests that the negatively-charged (native) Asp434 diminishes the ability of (negatively-charged) PAPS to occupy the site. The combined "positive" interactive effects of (e.g.) Arg515, Phe446, Phe529, Arg437, (etc.) and the "negative" effect of Asp434 delimits the concentration range at which intracellular PAPS can act as an effective allosteric inhibitor in vivo. This allows the
sulfonucleotide to accumulate to a level that is appropriate for the enzymes that use PAPS before inhibition sets in.
Impacts
Sulfur is one of the six major structural elements of living cells. Most of the inorganic sulfur on the earth's aerobic surface exists as ionic sulfate. It is in this form that the element enters living cells via the actions of ATP sulfurylase and APS kinase. Thus the sulfate activating enzymes play a role in sulfur metabolism analogous to those played by the carbon dioxide-fixing RuBP-carboxylase of photosynthetic organisms and nitrogenase of nitrogen-fixing organisms. An understanding of the molecular activities and structures of these enzymes are essential for any future protein engineering projects that may seek, for example, to (e.g.) improve the sulfur amino acid content of crops, or devise plant-based bioremediation methods for removing excess selenium from contaminated soils. (The sulfate activating enzymes do indeed accept selenate and APSe as substrates.) Of additional relevance -- ATP sulfurylase is a site of inhibition by chromate ("Cr-VI") and perchlorate,
two environmental pollutants. It is important to quantify the exact sensitivities of ATP sulfurylases from various sources to chromate.
Publications
- Zhihao, Y., Lansdon, E., Segel, I. H., and Fisher, A. J. 2007. Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aqufex aeolicus. J. Mol. Biol. 365, 732-743.
|
Progress 01/01/06 to 12/31/06
Outputs
Inorganic sulfate (from soil and water) serves as the sole sulfur source for most plants and aerobic microorganisms. Plants and microbes use sulfate to produce cysteine and methionine for protein biosynthesis and for the formation of several coenzymes such as CoA, biotin and thiamin. Animals use inorganic sulfate to form sulfate esters such as chondroitin sulfate, heparin, and sulfated hormones. Sulfate activation proceeds in two steps catalyzed by the sequential action of the enzymes ATP sulfurylase and APS kinase. The reactions produce, in order, the sulfonucleotides APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). Our recent research focused on the regulatory characteristics of ATP sulfurylase from P. chrysogenum, an enzyme that is allosterically inhibited by PAPS. X-ray crystallographic studies revealed that each monomer of the native hexameric enzyme is composed of three regions: an N-terminal domain, a central catalytic domain,
and a C-terminal (regulatory) domain. This last domain is evolutionarily derived from APS kinase and provides the binding site for PAPS. Structures with either APS or PAPS bound suggested that PAPS acts by disrupting a trans-triad salt link between Arg-515 in a C-terminal domain and Asp-111 in the N-terminal domain of a different monomer. It was hypothesized that this action triggers the concerted allosteric transition of the enzyme from a high proficiency R state to a lower proficiency T state. The R to T transition consists of a 27 degree rotation of each catalytic domain relative to an adjacent regulatory domain. Domain rotation is accompanied by the movement of a catalytic domain 'switch' which flips 'up' by 17 degrees. We believe that in the 'down' position, a switch residue (Asp-234) aligns MgATP and sulfate by neutralizing Arg-199 at the sulfate subsite. The hypothesis was investigated by site-directed mutagenesis experiments in which specific amino acids within the allosteric
domain were substituted. The mutants included Arg515Asp/Asp111Arg in which the salt link between positions 111 and 515 was preserved, but the positions of the partners were reversed. The kinetics results led to the following conclusions: (a) The 111-515 salt links stabilizes the R state of the enzyme. That is, the double mutant is perfectly stable and shows no cooperativity; But removal of either charged partner (e.g., as in Asp111Ala or Arg515Ala) allows a fraction of the enzyme molecules to attain the T state. (b) Position 515 must be occupied by a positively-charged residue in order to interact with the allosteric effector, PAPS. Thus Asp111Ala is quite sensitive to PAPS, but Arg515Ala and Asp111Arg/Arg515Asp show no increase in cooperativity in the presence of PAPS. All mutant enzymes, including those that were insensitive to PAPS were still able to assume the T state as induced by other (PAPS-independent) methods (e.g., higher temperature or lowered pH). Our future research is
aimed at establishing the exact sequence of molecular events by which the binding of the allosteric effector alters the catalytic efficiency of the active site.
Impacts
Sulfur is one of the six major structural elements of living cells. Most of the inorganic sulfur on the earth's aerobic surface exists as ionic sulfate. It is in this form that the element enters living cells via the actions of ATP sulfurylase and APS kinase. Thus the sulfate activating enzymes play a role in sulfur metabolism analogous to those played by the CO2-fixing RuBP-carboxylase of photosynthetic organisms and nitrogenase of N2-fixing organisms. An understanding of the molecular activities and structures of these enzymes are essential for any future protein engineering projects that may seek, for example, to (e.g.) improve the sulfur amino acid content of crops, or devise plant-based bioremediation methods for removing excess selenium from contaminated soils. (The sulfate activating enzymes do indeed accept selenate and APSe as substrates.) Of additional relevance - ATP sulfurylase is a site of inhibition by chromate ('Cr-VI') and perchlorate, two
environmental pollutants. It is important to quantify the exact sensitivities of ATP sulfurylases from various sources to chromate.
Publications
- No publications reported this period
|
Progress 01/01/05 to 12/31/05
Outputs
Inorganic sulfate (from soil and water) serves as the sole sulfur source for most plants and aerobic microorganisms. Animals use inorganic sulfate to form sulfate esters such as chondroitin sulfate, heparin, and sulfated hormones. Sulfate activation proceeds in two steps catalyzed, in order, by the enzymes ATP sulfurylase and APS kinase. The reactions produce the sulfonucleotides APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). Our recent research focused on the regulatory characteristics of ATP sulfurylase from P. chrysogenum, an enzyme that is allosterically inhibited by PAPS, the product of the APS kinase-catalyzed reaction. X-ray crystallographic studies revealed that each monomer of the native hexameric enzyme is composed of three regions: an N-terminal domain, a central catalytic domain, and a C-terminal (regulatory) domain.. This last domain is evolutionarily derived from APS kinase and provides the binding site for PAPS.
Structures with either APS or PAPS bound suggested that PAPS acts by disrupting a trans-triad salt link between Arg-515 in a C-terminal domain and Asp-111 in the N-terminal domain of a different monomer. Presumably, this action triggers the concerted allosteric transition of the enzyme from a high proficiency R state to a lower proficiency T state. The R to T transition consists of a 27 degrees rotation of each catalytic domain relative to an adjacent regulatory domain. Domain rotation is accompanied by the movement of a catalytic domain 'switch' which flips 'up' by 17 degrees. We believe that in the 'down' position, a switch residue (Asp-234) aligns MgATP and sulfate by neutralizing Arg-199 at the sulfate subsite. The primary trigger was confirmed by site-directed mutagenesis experiments in which either Arg-515 or Asp-111 was changed to Ala. The resulting mutant enzymes displayed intrinsic positive cooperativity. Our cumulative results suggest that domain rotation and restricted
switch movement may be part of the normal catalytic cycle and that PAPS exerts its allosteric inhibition by interfering with the amplitude of these motions. To better understand the roles of domain, loop, and subunit motions, we are currently constructing and kinetically characterizing several chimeric enzyme forms. These include an enzyme that has been engineered to contain true APS kinase in place of the normal, APS kinase-like C-terminal domain. This bifunctional protein may resemble an ancestral form that channeled APS between active sites. Other chimeras currently under study include (a) the N-terminal and catalytic domains of the allosteric P. chrysogenum enzyme joined to the C-terminal domain of the (non-allosteric) Saccharomyces ATP sulfurylase and (b) the N-terminal and catalytic domains of the yeast enzyme joined to the C-terminal domain of the P. chrysogenum enzyme. The latter may be useful in uncovering subunit motions during catalysis because preliminary examination shows
that it displays a slow structural transition when both substrates are bound. This transition probably also occurs in the normal wild type enzyme, but at a rate that is too rapid to detect by steady state kinetics.
Impacts
Sulfur is one of the six major structural elements of living cells. Most of the inorganic sulfur on the earth's aerobic surface exists as ionic sulfate. It is in this form that the element enters living cells via the actions of ATP sulfurylase and APS kinase. Thus the sulfate activating enzymes play a role in sulfur metabolism analogous to those played by the CO2-fixing RuBP-carboxylase of photosynthetic organisms and nitrogenase of N2-fixing organisms. An understanding of the molecular activities of these enzymes is essential for any future protein engineering projects that may seek, for example, to (e.g.) improve the sulfur amino acid content of crops, or devise plant-based bioremediation methods for removing excess selenium from contaminated soils. (The sulfate activating enzymes do indeed accept selenate and APSe as substrates.) Of additional relevance -- ATP sulfurylase is a site of inhibition by chromate ('Cr-VI') and perchlorate, two environmental pollutants.
It is important to quantify the exact sensitivities of ATP sulfurylases from various sources to chromate.
Publications
- No publications reported this period
|
Progress 01/01/04 to 12/31/04
Outputs
Inorganic sulfate serves as the sole sulfur source for most plants and aerobic microorganisms. Animals use inorganic sulfate to form important sulfate esters such as chondroitin sulfate, heparin, and several tyrosine-sulfated hormones. Because sulfate is non-reactive at cellular temperatures and pH, the anion must first be activated in order to enter the mainstream of metabolism. Activation proceeds in two steps. These are catalyzed, in order, by the enzymes ATP sulfurylase and APS kinase. The sequential reactions produce the sulfonucleotides APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). In most organisms, the two sulfate activating enzymes are separate proteins. But in animals, the two activities reside on a single polypeptide chain, i.e., in a single PAPS synthetase protein. In order to learn more about the catalytic properties of the bifunctional animal enzyme, and in particular, the functional relationship of the of the
N-terminal APS kinase domain to the downstream ATP sulfurylase domain, recombinant human PAPS synthetase, isoform 1 (brain) was purified to near-homogeneity from an E. coli expression system and kinetically characterized. The native enzyme is a dimer with each 71 kDa subunit containing an ATP sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (KI of 47.9 micromolar at saturating MgATP). The maximum attainable activity of 0.12 micromoles per min per mg protein was observed at an APS concentration ([APS]opt) of 15 micromolar. The theoretical Km for APS (at saturating MgATP) and the Michaelis constant for MgATP (at [APS]opt) were 4.2 micromolar 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 micro moles per min per mg protein. Upon addition of pure P. chrysogenum APS kinase in excess, the overall rate increased to 0.47 micro moles per min per mg protein. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) the accumulation of APS in solution during the overall reaction, (the rate acceleration provided by exogenous APS kinase, and (d) the 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 PAPSS1.the kinetic properties of recombinant human brain ATP sulfurylase.
Impacts
Our research has shown that (a) a regulatory region of an enzyme can evolve from a pre-existing enzyme, (b) a regulatory response might be fashioned from an enzyme conformation that is already part of the normal catalytic cycle, and (c) that a regulatory region can evolve to have new functions in promoting the catalytic reaction. These ideas are useful concepts for the future engineering of enzymes for specific tasks.
Publications
- Fisher, A. J., MacRae, I. J., Beynon, J. D., Lansdon, E. B., and Segel, I. H. Optimizing an enzyme for its physiological role: Structural and functional comparisons of ATP sulfurylase from three different organisms. In Conformational Proteomics of Macromolecular Architecture . World Scientific. N.J. Ch. 11 pp 222-241 (2004).
- Segel, I. H. Enzyme Kinetics. (Invited article.) Encyclopedia of Biological Chemistry. Elsevier. Vol. 2, pp 38-44) (2004).
- Hanna, E., Ng, K. F., MacRae, I. J., Bley, C. J., Fisher, A. J., and Segel, I. H. Kinetic and Stability Properties of P. chrysogenum ATP sulfurylase missing the C-terminal regulatory domain. J. Biol. Chem. 279, 4415-4424 (2004).
- Lansdon, E. B., Fisher, A. J., and Segel, I. H. Human PAPS Synthetase (Isoform 1; Brain): Kinetic Properties of the ATP Sulfurylase and APS Kinase Domains. Biochemistry 43, 4356-4365 (2004).
|
Progress 01/01/03 to 12/31/03
Outputs
Most plants and microorganisms can use inorganic sulfate as their sole source of sulfur. Because sulfate is nonreactive at cellular temperatures and pH, the anion must first be 'activated' in order to enter the mainstream of metabolism. Activation proceeds in two steps. These are catalyzed, in order, by the enzymes ATP sulfurylase and APS kinase. The sequential reactions produce the sulfonucleotides APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). ATP sulfurylase from the filamentous fungus, Penicillium chrysogenum, is a homooligomer composed of six 63.7 kDa subunits. PAPS is an allosteric inhibitor. This inhibition may be part of a sequential feedback process considering that PAPS is a major branch point metabolite in filamentous fungi, but not in other organisms. P. chrysogenum ATP sulfurylase is organized as a dimer of triads. Each subunit is composed of three structurally distinct globular regions: Residues 1-170 compose a
distinct N-terminal domain. Residues 171-395 compose the central catalytic domain. The allosteric site is located in a C-terminal domain that is very similar to APS kinase in sequence and structure. However, this regulatory domain (residues 396-573) has no APS kinase activity because of modifications to the ATP P-loop and the filling of the ATP binding region with protein side chain surrogates. PAPS is believed to initiate the allosteric transition by disrupting a salt link between Arg-515 in the C-terminal domain of one subunit and Asp-111 in the N-terminal domain of a trans-triad subunit. The R to T transition is accompanied by the movement of a catalytic domain loop (residues 228-238, termed the active site switch), which flips 'up' by 17 angstrom. When the switch is in the closed position, Asp-234 interacts with and presumably modulates the charge on Arg-199 of the site sulfate/phosphosulfate binding site. In order to learn more about the allosteric transition, and particularly,
more about the functional relationship of the of the C-terminal domain to the rest of the protein, the properties of recombinant P. chrysogenum ATP sulfurylase missing residues 396-573 have been examined. The results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E ATP and E sulfate complexes, and preceding the release of the first product (pyrophosphate) is optimized. The more negative entropy of activation of the truncated enzyme for the APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.
Impacts
Our research has shown that (a) a regulatory region of an enzyme can evolve from a pre-existing enzyme, (b) a regulatory response might be fashioned from an enzyme conformation that is already part of the normal catalytic cycle, and (c) that a regulatory region can evolve to have new functions in promoting the catalytic reaction. These ideas are useful concepts for the future engineering of enzymes for specific tasks.
Publications
- Fisher, A. J., MacRae, I. J., Beynon, J. D., Lansdon, E. B., and Segel, I. H. 2003. Optimizing an enzyme for its physiological role: Structural and functional comparisons of ATP sulfurylase from three different organisms. Ch. 10 in Proceedings of Nobel Forum on Structural Biology. Karolinska Institute.
- Segel, I. H. 2004. Enzyme Kinetics. (Invited article.) Encyclopedia of Biological Chemistry. Elsevier. In press.
- Hanna, E., Ng, K. F., MacRae, I. J., Bley, C. J., Fisher, A. J., and Segel, I. H. 2003. Kinetic and Stability Properties of P. chrysogenum ATP sulfurylase missing the C-terminal regulatory domain. J. Biol. Chem. In press.
|
Progress 01/01/02 to 12/31/02
Outputs
In sulfate assimilators, the physiological function of the enzyme ATP sulfurylase is to produce adenosine 5'-phosphosulfate (APS) from inorganic sulfate and ATP. However, in sulfur chemolithotrophs, the physiological reaction is in the opposite direction, i.e., the enzyme catalyzes the pyrophosphorolysis of APS forming ATP and sulfate. Recent research in our laboratory focused on the properties of ATP sulfurylase from Aquifex aeolicus, a bacterium isolated from a hot volcanic vent. The bacterium can use molecular hydrogen or reduced inorganic sulfur as an energy source (electron donor) and oxygen as the terminal electron acceptor. We were drawn to the ATP sulfurylase of A. aeolicus for several interrelated reasons. First, there is a scarcity of information about the kinetic properties of the enzyme from chemolithotrophs. In particular, we wanted to explore the hypothesis that certain kinetic properties of ATP sulfurylase are optimized for the physiological direction.
Second, there was the likelihood that the A. aeolicus enzyme would be highly heat stable and thus, it could serve as a subject for future x-ray crystallography investigations aimed at deciphering the structural basis of temperature optimization. Third, at about 44% overall identity to P. chrysogenum ATP sulfurylase, the A. aeolicus enzyme is the closest bacterial ortholog to ATP sulfurylases from filamentous fungi. More intriguingly, the domain order of A. aeolicus ATP sulfurylase is also identical to that of the fungal enzyme. That is, both the P. chrysogenum and A. aeolicus subunits contain a distinct N-terminal domain (A. aeolicus residues 1-142), a central catalytic domain (residues 143 - 304), and a C-terminal domain (residues 372 - end). The sequence of the C-terminal domain is about 40% identical to that of APS kinase, the second enzyme in the sulfate activation pathway of sulfate assimilators. Kinetic evidence indicated that the C-terminal domain of fungal ATP sulfurylase
provides a regulatory site for the allosteric inhibitor, 3-phosphoadenosine 5'-phosphosulfate (PAPS). In contrast, the C-terminal domain of the A. aeolicus ATP sulfurylase displayed APS kinase activity. This indicates that under appropriate environmental conditions, the enzyme might function in sulfate assimilation. Our studies also established that the Aquifex enzyme is highly heat stable with a half-life > 1 hr at 90 degrees C. The steady state kinetics are consistent with a mechanism where ATP and sulfate add randomly, but pyrophosphate is released before APS. The kinetic constants suggest that the enzyme is optimized to act in the direction of ATP + sulfate formation. Calculations showed that the ATP sulfurylase of chemolithotrophs provides an efficient route for recycling all of the pyrophosphate produced by biosynthetic reactions. The Aquifex enzyme may also function to produce PAPS for sulfate ester formation or sulfate assimilation when hydrogen serves as the energy source and
a reduced inorganic sulfur source is unavailable.
Impacts
Our research has shown (a) that a regulatory region of an enzyme can evolve from a pre-existing enzyme and (b) that a regulatory response might be fashioned from an enzyme conformation that is already part of the normal catalytic cycle. These ideas are useful concepts for the future engineering of enzymes for specific tasks.
Publications
- Beynon, J. D., MacRae, I. J., Huston, S. L., Nelson, D. C., Segel, I. H., and Fisher, A. J. Crystal Structure of ATP Sulfurylase from the Bacterial Symbiont of Riftia pachyptila, the Hydrothermal Vent Tubeworm. Biochemistry 40, 14509-14517 (2001).
- Hanna, E., MacRae, I. J., Medina, D., Fisher, A. J., and Segel, I. H. ATP Sulfurylase from the Hyperthermophilic Chemolithotroph Aquifex aeolicus. Arch. Biochem. Biophys. 406, 275-288 (2002).
- MacRae, I. J., Segel, I. H., and Fisher, A. J. Allosteric inhibition via R-state destabilization in ATP sulfurylase from P. chrysogenum. Nature Structural Biology 9, 945-949 (2002).
- Lansdon, E. B., Segel, I. H., and Fisher, A. J. Ligand-induced structural changes in adenosine 5'-phosphosulfate (APS) kinase from Penicillium chrysogenum. Biochemistry 41, 13672-13680 (2002).
|
Progress 01/01/01 to 12/31/01
Outputs
The effects of changing temperature on the initial velocity kinetics of allosteric ATP sulfurylase from P. chrysogenum were measured using the molybdolysis assay. (Molybdate is an alternative substrate that substitutes for inorganic sulfate.) The experiments were prompted by the following properties of the fungal sulfate activating enzymes: (a) The normal allosteric effector of fungal ATP sulfurylase is PAPS (3-phosphoadenosine-5-phosphosulfate), a product of the enzyme APS kinase. (b) The C-terminal regulatory domain of fungal ATP sulfurylase (which provides the binding site for PAPS) is nearly identical in structure to APS kinase. (c) True APS kinase is a homodimer that undergoes a temperature-dependent, reversible dissociation of subunits over a narrow temperature range. (d) The regulatory domains of fungal ATP sulfurylase also form intersubunit "dimers". Therefore, if the C-terminal domains of fungal ATP sulfurylase retain the APS kinase-like ability to undergo
reversible separation, and this movement participates in the allosteric transition (normally induced by PAPS), changing the temperature (in the absence of PAPS) might have a systematic effect on cooperativity. Experimentally, wild type ATP sulfurylase from P. chrysogenum yielded hyperbolic initial velocity curves between 18 and 30 C. Increasing the assay temperature above 30 C at a constant pH of 8.0 increased the cooperativity of the velocity curves. Hill coefficients (nH) up to 1.8 were observed at 42 C. The characteristics of the bireactant kinetics at 42 C were the same as those observed at 30 C in the presence of PAPS. In contrast, yeast ATP sulfurylase (which has a highly degenerate C-terminal domain but is otherwise almost identical in structure to the P. chrysogenum enzyme) yielded hyperbolic plots at 42 C. The P. chrysogenum mutant enzyme, C509S, which is intrinsically cooperative (nH = 1.8) at 30 C, became more cooperative as the temperature was increased; nH for varied
MgATP at 5 mM molybdate was 2.2 at 37 C and 2.9 at 42 C. As the temperature was decreased, the cooperativity of C509S decreased; nH was 1.0 at 18 C. The cumulative results indicate that increasing the temperature promotes a shift in the base level distribution of enzyme molecules from the R state toward the T state (i.e., increases the allosteric constant, L.) As a result, the enzyme displays a true temperature optimum at subsaturating MgATP. Movement of C-terminal domains about their "dimer" interface is a possible participant in the allosteric transition. The reversible temperature-dependent transitions of fungal ATP sulfurylase and APS kinase may play a role in energy conservation at high temperatures where the organism can survive, but not grow optimally.
Impacts
Our investigation of the effect of temperature on the kinetic characteristics of P. chrysogenum ATP sulfurylase activity disclosed a rare example of a true temperature optimum of an enzyme. Unlike most previous reports of the so-called "optimum temperature" the underlying model proposed for the P. chrysogenum enzyme does not involve irreversible inactivation. Consequently, the "optimum temperature" in this case is an intrinsic constant that is not dependent upon the assay time.
Publications
- Roy, H., Diwan, J., Segel, L. D., and Segel, I. H. Computer-assisted simulations of phosphofructokinase-1 kinetics using simplified velocity equations. Biochemistry and Molecular Biology Education. 29, 3-9 (2001).
- MacRae, I. J., Segel, I. H., and Fisher, A. J. Crystal Structure of ATP Sulfurylase from Penicillium chrysogenum: Insights into the allosteric regulation of sulfate assimilation. Biochemistry 40, 6795-6804 (2001).
- Medina, D., Hanna, E., MacRae, I. J., Fisher, A. J., and Segel, I. H. Temperature Effects on the Allosteric Transition of ATP Sulfurylase from Penicillium chrysogenum. Arch. Biochem. Biophys. 293, 51-60 (2001).
|
Progress 01/01/00 to 12/31/00
Outputs
ATP sulfurylase from P. chrysogenum is a homohexameric allosteric enzyme in which Cys-509 is critical for maintaining the R state. Cys-509 is located in a C-terminal domain that is homologous to APS kinase, the second enzyme of the sulfate assimilation pathway. Replacement of Cys-509 with either Tyr or Ser destabilizes the R state resulting in an active sulfurylase that is intrinsically cooperative at pH 8 in the absence of the allosteric effector, 3-phosphoadenosine 5-phosphosulfate (PAPS). However, the kinetics of C509Y and C509S are not identical. Velocity curves of C509Y (varied MgATP or MoO42-) display an increasing Hill coefficient, nH, as the concentration of the non-varied substrate is increased. In contrast, the v versus [MgATP] plots of C509S have a near-constant nH (ca. 2) at all [molybdate]; the nH of the v versus [molybdate] plots decrease with increasing [MgATP], approaching 1.0 at saturating MgATP. The results suggest that in the absence of ligands,
C509Y is driven further toward the T state than is C509S -- a conclusion that is supported by the opposite effects of changing MgATP concentration on the activation of the two mutant enzymes by inorganic thiosulfate, an inhibitor competitive with molybdate (or sulfate). The opposite effects of PAPS on the Hill coefficient --increasing the nH of C509S to 2.9, but decreasing nH of C509Y -- also supports this conclusion. The bireactant kinetics of C509Y are similar to those of the wild type enzyme in which Cys-509 has been covalently modified. The kinetics of C509S are similar to those of the wild type enzyme in the presence of PAPS. The cumulative results suggest that the negatively charged side chain of Cys-509 plays a role in stabilizing the R state. Protonating Cys-509 does indeed induce cooperativity. But this can not be the whole story because the Hill coefficient of C509S also increases as the pH is decreased. His residues (His-508?) may also play an important role. In true APS
kinase, the residue analogous to Cys-509 is located in a short helix that immediately precedes a mobile loop. This loop (which contains a putative PAPS binding motif and a "quick trypsin" site) is a hinged element ("ATP lid") that immobilizes and protects bound MgATP during catalysis by APS kinase. The analogous "quick trypsin" site of ATP sulfurylase is more accessible in the mutant enzymes than in the wild-type enzyme suggesting that movement and exposure of the mobile loop occurs during the R to T transition. It is not unexpected then to find that amino acid replacements within the helix (hinge?) alter the allosteric equilibrium.
Impacts
Sulfur is one of the six most abundant elements found in living cells. The enzymes that we study catalyze the first two reactions by which inorganic sulfate (from soil, or water) is incorporated into organic molecules. Our experiments provide insight into the mechanisms by which these enzymes are regulated and the evolution of allosteric sites
Publications
- MacRae, I. J., Segel, I. H., and Fisher, A. J. (2000). Crystal Structure of adenosine 5'- phosphosulfate (APS) Kinase from P. chrysogenum. Biochemistry 39, 1613-1621.
- DeWolf, W., Jr. and Segel, I. H. (1999). A Simplified Velocity Equation for Characterizing the Partial Inhibition or Nonessential Activation of a Bireactant Enzyme. J. Enzyme Inhibition. 15, 311-333 (2000).
- MacRae, I. J., Hanna, E., Ho, J. D., Fisher, A. J., and Segel, I. H. (1999). Induction of positive cooperativity by amino acid replacements within the C-terminal domain of P. chrysogenum ATP sulfurylase. J. Biol. Chem. 275, 36303-36310 (2000).
|
Progress 01/01/99 to 12/31/99
Outputs
Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the overall activation of inorganic sulfate viz., the ATP-dependent formation of 3'phosphoadenosine-5'-phosphosulfate (PAPS) from APS. The enzyme is unusual in that it is subject to strong inhibition by its own substrate, APS. It appears that APS kinases from different sources have different mechanisms of substrate inhibition. The inhibition has been variously reported to be uncompetitive with respect to MgATP (resulting from the formation of a dead end E APS MgADP complex) or competitive with MgATP (resulting from the formation of a dead end E.APS complex). Theoretical and experimental analyses have shown that these two types of substrate inhibition can be differentiated for ordered kinetic mechanisms. A key diagnostic feature is that, at any given inhibitory level of APS, enzyme activity relative to the velocity at [APS]opt (v/vopt) decreases as the fixed [MgATP] is increased in the
ncompetitive system, while in the competitive system the relative activity increases as the fixed [MgATP] is increased. Normalized plots of v/vopt versus [APS] clearly display these distinguishing characteristics. The method confirmed that P. chrysogenum APS kinase exhibits uncompetitive inhibition by APS. Preliminary studies on the x-ray structure of the enzyme are consistent with the proposed mechanism of substrate inhibition.
Impacts
The new method allows investigators to distingush between the two major types of substrate inhibition by inspection of the v versus [inhibitory substrate] plots at different fixed concentrations of the non-inhibitory substrate. Linear diagnostic plots are unnecessary. The approach will be useful to investigators who are reluctant to perform a complete kinetic analysis of substrate inhibition data.
Publications
- MacRae, I. and Segel, I. H. Adenosine 5'-phosphosulfate (APS) Kinase: Diagnosing the Mechanism of Substrate Inhibtion. Arch. Biochem. Biophys. 361, 277 - 282 (1999). ASSOCIATED COVER ART: 361, Nos. 1 and 2 (1999).
|
Progress 01/01/98 to 12/01/98
Outputs
The properties of Penicillium chrysogenum adenosine 5'-phosphosulfate (APS) kinase mutated at ser-107 were examined. Ser-107 is analogous to a serine of the E. coli enzyme which has been shown to serve as an intermediate acceptor in the transfer of a phosphoryl group from ATP to APS. Replacement of ser-107 with alanine yielded an active enzyme with kinetic characteristics similar to those of wild-type APS kinase. Another mutant form of the enzyme in which ser-107 was replaced by cysteine was also active. Covalent modification of cys-107 eliminated catalytic activity while substrates protected against modification. Mutation of ser-97, or ser-99, or thr-103, or ser-104 to alanine, or of tyr-109 to phenylalanine also yielded an active enzyme. The cumulative results indicate that ser-107 may reside in the substrate -binding pocket of fungal APS kinase, but neither it nor any nearby hydroxy amino acid serves as an obligatory phophoryl acceptor in the PAPS synthesis
reaction. The results also indicate that the absence of a serine at position 478 in the APS kinase-like C-terminal region of fungal ATP sulfurylase does not account for the lack of APS kinase activity in that enzyme. However, mutating the ATP P-loop residues in APS kinase to those found in the analogous C-terminal region of fungal ATP sulfurylase eliminated enzyme activity.
Impacts
(N/A)
Publications
- MACRAE, I., ROSE, A.B. and SEGEL, I.H. 1998. Adenosine 5'-Phosphosulfate (APS) Kinase from Penicillium chrysogenum: Site Directed Mutagenesis Studies on Putative Phosphoryl-Accepting and ATP P - Loop residues. J. Biol. Chem. 273: 28583.
|
Progress 01/01/95 to 12/30/95
Outputs
APS kinase catalyzes the second reaction in the biological activation of inorganic sulfate. We have identified active site residues by a differential labeling method: The enzyme was modified by fluorescein-5-isothiocyanate (FITC) in the absence and in the presence of MgADP + APS. After tryptic proteolysis, the "protected" vs. "unprotected" HPLC peptide spectra were compared, and outstanding peptides isolated. Three peptides stood out as being markedly different in digests of protected versus unprotected labeled enzyme. One was gly-leu-tyr-lys-lys. This sequence corresponds to residues 152-156 which includes the gly of the smaller "ATP motif" (DPKG). The second fluorescent peptide corresponded to residues 159-178, which includes a portion of the putative PAPS motif, KAREGVIKEFTG. In this peptide, lys-163 was modified. The third outstanding peptide was non-fluorescent and more abundant in digests of the enzyme modified in the presence of saturating nucleotides. The
sequence corresponded to residues 39-51 which immediately follows the larger "ATP P-loop motif', GLSASGK. Our above results provide the first chemical evidence supporting the role of the PAPS motif in the binding of phosphosulfate nucleotides to APS kinase. The significance of the motif was unclear; it is present in APS kinases and in many sulfotransferases, but it does not appear in PAPS reductase.
Impacts
(N/A)
Publications
|
Progress 01/01/94 to 12/30/94
Outputs
ATP sulfurylase catalyzes the first intracellular reaction in the incorporation of inorganic sulfate into organic molecules by sulfate assimilating organisms. Fungal and yeast ATP sulfurylases have similar kinetic and chemical properties except that the fungal enzyme (a) contains a reactive cys residue (SH-1) whose modification results in sigmoidal velocity curves and (b) is allosterically inhibited by PAPS. Also, the fungal enzyme subunit (MW 63 kDa) is larger than that of the yeast enzyme (MW 57 kDa). To correlate the allosteric properties of the fungal enzyme with specific structural features, we cloned and sequenced ATP sulfurylase from P. chrysogenum. The yeast and fungal enzymes are homologous over the first 400 amino acids and contain two basic regions which are conserved in sulfurylases from plants and chemolithotrophic bacteria. These basic regions may participate in forming the substrate binding site. There is no significant sequence homology between the
yeast and fungal enzymes in the C-terminal 171 amino acids (where SH-1 resides). However there is significant homology between this domain and a portion of APS kinase, the next enzyme in the pathway (which forms PAPS). The cumulative results suggest that (a) the PAPS binding site of P. chrysogenum ATP sulfurylase is located in the C-terminal domain of the enzyme and (b) this domain is derived from an ancestoral APS kinase.
Impacts
(N/A)
Publications
|
Progress 01/01/93 to 12/30/93
Outputs
ATP sulfurylase catalyzes the synthesis of adenosine-5-phosphosulfate (APS), which is the first intracellular reaction in the incorporation of inorganic sulfate into organic molecules by sulfate assimilating organisms. The kinetic properties of fungal and yeast ATP sulfurylases are very similar except that the former (a) contains a reactive cys residue whose modification results in sigmoidal velocity curves and (b) is allosterically inhibited by PAPS. In an attempt to correlate the unique properties of the fungal enzyme with specific structural features, we compared the primary sequences of the S. cereviseae and P. chrysogenum enzymes. Our results can be summarized as follows: (a) The sequence of the fungal enzyme is homologous to that of the yeast enzyme (54 percent identity in 517 amino acids). (b) Fungal - yeast sequence homology disappears beyond residue 400. (c) Both enzymes contain two highly conserved regions. The first of these stretches is identical in 17-19
out of 21 residues and contains 5-6 basic residues, making it a candidate for participation in forming the substrate binding site. (d) The fungal enzyme is larger than the yeast enzyme by ca. 100 residues, most of which are located in the C-terminal region. The extra amino acid residues account for the size difference between the yeast enzyme subunit (ca. 60 kDa) and the fungal enzyme subunit (ca. 63-66 kDa).
Impacts
(N/A)
Publications
|
Progress 01/01/92 to 12/30/92
Outputs
ATP sulfurylase catalyzes the first reaction in the biological incorporation of inorganic sulfate. The enzyme from P. CHRYSOGENUM, A. NIDULANS, and N. CRASSA contains a reactive cysteinyl residue (SH-1). IN VITRO modification of SH-1 results in increased S0.5 values for MgATP and S042- and alters the kinetics from hyperbolic to sigmoidal. No evidence was obtained for SH-1 modification IN VIVO when the need for sulfate activation was diminished. However, in the presence of PAPS, (the product of APS kinase, the second enzyme of the two-step activation sequence), the initial velocity plots of the unmodified enzyme become sigmoidal and the S0.5 values increase. The results suggest that IN VITRO SH-1 modification induces a conformation in the enzyme that is identical to that formed IN VIVO by the reversible binding of PAPS. ATP sulfurylases from yeast, plant leaves, and rata liver do not possess an SH-1-like cysteinyl residue, do not display sigmoidal kinetics in the
presence of PAPS, and are much less sensitive to inhibition by PAPS compared to the fungal enzymes. Experiments are in progress to clone the ATP sulfurylase gene with the objective of replacing the allosteric cysteine residue (by site-directed mutagenesis) with other amino acids. We hope to establish the chemical function of the allosteric site sulfhydryl group.
Impacts
(N/A)
Publications
|
Progress 01/01/86 to 12/30/91
Outputs
ATP sulfurylase catalyzes the first reaction in the biological incorporation of inorganic sulfate. The enzyme from P. CHRYSOGENUM, A. NIDULANS, and N. CRASSA contains a reactive cysteinyl residue (SH-1). IN VITRO modification of SH-1 results in increased (S)0.5 values for MgATP and SP42- and alters the kinetics from hyperbolic to sigmoidal. No evidence was obtained for SH-1 modification IN VIVO when the need for sulfate activation was diminished. However, in the presence of PAPS, (the product of APS kinase, the second enzyme of the two-step activation sequence), the initial velocity plots of the unmodified enzyme become sigmoidal and the (S)0.5 values increase. The results suggest that IN VITRO SH-1 modification induces a conformation in the enzyme that is identical to that formed IN VIVO by the reversible binding of PAPS. ATP sulfurylases from yeast, plant leaves, and rata liver do not possess an SH-1-like cysteinyl residue, do not display sigmoidal kinetics in
the presence of PAPS, and are much less sensitive to inhibition by PAPS compared to the fungal enzymes. (35S) Adenosine-5'-phosphosulfate (APS) binding to P. CHRYSOGENUM APS kinase was measured by centrifugal ultrafiltration. The results support our conclusion that substrate inhibition of the fungal enzyme by APS results from the formation of a dead end E.MGADP.APS complex. That is, APS binds to the subsite vacated by PAPS in the compulsory (or predominately) ordered product release sequence (PAPS before MgADP).
Impacts
(N/A)
Publications
|
Progress 01/01/90 to 12/30/90
Outputs
ATP sulfurylase catalyzes the first reaction in the biological incorporation of inorganic sulfate. The enzyme from P. CHRYSOGENUM, A. NIDULANS, and N. CRASSA contains a reactive cysteinyl residue (SH-1). IN VITRO modification of SH-1 results in increased ?S?0.5 values for MgATP and S042- and alters the kinetics from hyperbolic to sigmoidal. No evidence was obtained for SH-1 modification IN VIVO when the need for sulfate activation was diminished. However, in the presence of PAPS, (the product of APS kinase, the second enzyme of the two-step activation sequence), the initial velocity plots of the unmodified enzyme become sigmoidal and the ?S?0.5 values increase. The results suggest that IN VITRO SH-1 modification induces a conformation in the enzyme that is identical to that formed IN VIVO by the reversible binding of PAPS. ATP sulfurylases from yeast, plant leaves, and rata liver do not possess an SH-1-like cysteinyl residue, do not display sigmoidal kinetics in the
presence of PAPS, and are much less sensitive to inhibition by PAPS compared to the fungal enzymes. ?35S? Adenosine-5'-phosphosulfate (APS) binding to P. CHRYSOGENUM APS kinase was measured by centrifugal ultrafiltration. APS did not bind to the free enzyme with a measurable affinity even at low ionic strength where substrate inhibition by APS is quite marked. However, APS bound with an apparent Kd of 0.54 micromoles in the presence of 5 mM MgADP.
Impacts
(N/A)
Publications
|
Progress 01/01/89 to 12/30/89
Outputs
ATP sulfurylase catalyzes the first reaction in the biological incorporation of inorganic sulfate. The enzyme from P. chrysogenum, A. nidulans, and N. crassa contains a reactive cysteinyl residue (SH-1). In vitro modification of SH-1 results in increased (S(0.5 values for MgATP and SO(4) and alters the kinetics from hyperbolic to sigmoidal. No evidence was obtained for SH-1 modification in vivo when the need for sulfate activation was diminished. However, in the presence of PAPS, (the product of the second enzyme of the two-step activation sequence), the initial velocity plots of the unmodified enzyme become sigmoidal and the (S)(0.5) values increase. The results that in vitro SH-1 modification induces a conformation in the enzyme that is identical to that formed in vivo by the reversible binding of PAPS. ATP sulfurylases from yeast, plant leaves, and rat liver do not possess an SH-1-like cysteinyl residue, do not display sigmoidal kinetics in the presence of
PAPS, and are much less sensitive to inhibition by PAPS compared to the fungal enzymes.
Impacts
(N/A)
Publications
|
Progress 01/01/88 to 12/30/88
Outputs
Results have shown that ATP sulfurylase from Penicillium chrysogenum is a homohexamer that contains three free sulfhydryl groups per subunit; one of which (SH-1) can be modified by specific reagents under non-denaturing conditions. Modification of SH-1 causes the velocity curves become sigmoidal with a Hill coefficient (n(subscript H)) of 2. Equilibrium binding measurements confirmed that ?(superscript 32)P?MgATP binds to the SH-modified enzyme in a positively cooperative fashion if a sulfate subsite ligand is present. ?(superscript 35)S?APS binding to the SH-modified enzyme displays positive cooperativity (n(subscript H) = 1.9) in the absence of a PP(subscript i) subsite ligand. Indicating that positive cooperativity requires occupancy of the adenylyl and sulfate (but not the pyrophosphate) subsites. ?(superscript 35)S?APS binding to the native enzyme displays negative cooperativity. Isotope trapping profiles for the single turnover of ?(superscript 35)S?APS confirm
the equilibrium binding curves, indicate that all six sites per hexamer are catalytically active, and show that APS does not dissociate at a significant rate from E.APS.PP(subscript i). Cumulative data suggest a model in which pairs of sites or subunits can exist in three different states designated HH (both sites have a high APS affinity, as in the native free enzyme) LL (both sites have a low APS affinity as in the SH-modified enzyme) and LH (as in the APS-occupied native or SH-modified enzyme).
Impacts
(N/A)
Publications
|
Progress 01/01/87 to 12/30/87
Outputs
ATP sulfurylase from P. chrysogenum contains three free sulfhydryl groups per subunit. Under non-denaturing conditions, only one SH group per subunit (designated SH-1) is modified by DTNB, NEM, etc. Modification of SH-1 had only a small effect on k(cat), but markedly increased the (S)(0.5) values for the substrates, MgATP and SO(4). The SH-modified enzyme displayed sigmoidal velocity curves for both substrates with Hill coefficients (n(H)) of 2. In order to determine whether the sigmoidicity resulted from true cooperative binding, the shapes of the binding curves were established from the degree of protection provided by a ligand against phenylglyoxal-dependent irreversible inactivation under equilibrium, non-catalytic conditions. Both the native and the SH-modified enzyme displayed hyperbolic plots of delta k (i.e., protection) versus (MgATP), or (FSO(3)), or (S(2)O(3)) in the absence of coligand. The plots of delta k versus (ligand) for the native enzyme were
also hyperbolic in the presence of a fixed concentration of coligand (n(H) = 0.98 +/- 0.06). However, in the presence of a fixed (FSO(3)) or (S(2)O(3)), the delta k versus (MgATP) plot for the SH-modified enzyme was sigmoidal, as was plot of delta k versus (FSO(3)-) or (S(2)O(3)) inhe presence of fixed (MgATP). The cumulative n(H) values were 1.92 +/- 0.09. The results indicated that substrates (or analogs) bind hyperbolically to unoccupied SH-modified subunits, but in a subunit-cooperative fashion to form a ternary complex.
Impacts
(N/A)
Publications
|
Progress 01/01/86 to 12/30/86
Outputs
In order to fully understand the role of inorganic pyrophosphate (PP(i)) in energy metabolism and metabolic control, we must know its steady state intracellular concentration and its phosphoryl transfer potential. It occurred to us that the enzyme ATP sulfurylase could be used to measure low concentrations of PP(i). Accordingly, a continuous, coupled, spectrophotometric assay was developed in which ATP sulfurylase was employed to measure the concentration of PP(i) at equilibrium with known concentrations of inorganic orthophosphate (P(i)) in the presence of excess PP(i)tase. The apparent equilibrium constant (K(eq,app)) of the PP(i) hydrolysis reaction was shown to decrease as the concentration of Mg is increased. At pH 7.3, 30C, in the presence of 150 mM NaC1 and 1 mM free Mg, K(eq,app) (calculated as (P(i))(t)/(PP(i))(t)) was 1950. A radiochemical end point assay for measuring unknown concentrations of PP(i) was also devised. In the presence of excess
S-adenosine-5'-phosphosulfate (S-APS) as the cosubstrate, SO(4) formation was stoichiometric with added PP(i). In the absence of interfering substances, as little as 2 pmoles of PP(i) per m1 assay volume can be measured. The cellular PP(i) and P(i) level of several animal, plant, and fungal tissues was determined. The results showed that the PP(i)tase reaction was markedly displaced from equilibrium in vivo lending credance to the hypothesis that PP(i) is a regulator of nucleotidyl transferase reactions.
Impacts
(N/A)
Publications
|
Progress 01/01/85 to 12/30/85
Outputs
By the judicious use of dye ligand chromatography, we succeeded in separating APS kinase from ATP sulfurylase. We also perfected a sensitive pyruvate kinase + lactate dehydrogenase-coupled assay which allowed us to complete the purification of APS kinase and characterize the kinetics of the reaction as obligately dordered: MgATP adds before APS; PAPS dissociates before MgADP. The potent substrate inhibition by APS was traced to the formation of an "abortive" (dead end) EmgADPAPS complex. In another study, we showed by kinetic and physical techniques that the reversible heat inactivation of APS kinase resulted from subunit dissociation and reassociation. In our latest study on APS kinase we confirmed the formation of the EMgADPAPS complex by analyzing the protection provided by mixtures of MgADP and APS against lysyl side chain modification. Monohydroxy bile salts are toxic to the liver. Animals modify the bile salts by sulfation, which enhances excretion. In a
collabotative project with Dr. Lee Chen (Department of Internal Medicine, UCD School of Medicine), the single isozyme bile salt sulfotransferase of normal human male liver was partially purified and characterized. This work established a base line for future studies on possible sulfotransferase isozyme patterns of human female liver (the female rat produces two distinct bile salt sulfotransferase isozymes) and the effect of various liver disease states on the levels or amounts of bile salt detoxifying enzymes.
Impacts
(N/A)
Publications
|
Progress 01/01/84 to 12/30/84
Outputs
The kinetics of the forward ATP sulfurylase-catlayzed reaction were examined using a new assay based on DTyPP(i) released from gamma-DTyP-MgATP in the presence of inorganic sulfate. Replots yielded Vmax(f) = 6.6 units x mg proteinEPG, Km(A) = 0.13 mM, K(ia) = 0.33 mM, and Km(B) = 0.55 mM where A = MgATP and B = SO2-/4. Thiosulfate, a dead end inhibitor of the reaction, was competitive with sulfate and noncompetitive with respect to MgATP. The ratio k(cat)/Km(A) was determined for several alternative inorganic substrates, B, where A = MgATP and B = SO2-/4, SeO2-/4, MoO2-/4, WO2-/4, or CrO2-/4. For SO2-/4 and SeO2-/4, the ratio was 5 - 6.5, x 10, MEPG x secEPG; for the others, the ratio was 5.8 - 7.3 x 101 MEPG x secEPG. If the reaction with each alternative B subtrate was ordered with MgATP adding first, the ratio kcat/Km(A) would be constant and equivalent to k(on) for MgATP binding. The kinetics of the reverse reaction were examined using a new assay based on DT1SOyE
release from DT1S-APS in the presence of MgPP(i). Reciprocal plots were linear, intersecting below the horizontal axis. Replots yielded Vmax(r) = 50 units x mg protein EPG, Km(Q) = 0.3 mu M, K(iq) = 0.04 mu M, and Kmp = 4 mu M where Q = Q = APS and P = PP(i) (total of all species). MgATP and SO2-/4 were both competitive with APS and noncompetitive with respect to MgPP(i).
Impacts
(N/A)
Publications
|
Progress 01/01/83 to 12/30/83
Outputs
Adenosine-5'-phosphosulfate (APS) kinase, the second enzyme in the pathway of inorganic sulfate assimilation, was purified to near homogeneity from mycelium of the filamentous fungus, Penicillium chrysogenum. The enzyme has a native molecular weight of 60,000 and is composed of two 30,000 dalton subunits. The most highly purified preparation has a specific activity of 24.7 units x mg protein EPG in the physiological direction of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) formation. This activity is nearly 100-fold higher than that of any previously purified preparation. The steady-state kinetics of the reaction were investigated. The cumulative results suggest that the kinetic mechanism is ordered in both directions with MgATP adding before APS, and PAPS leaving before MgADP. The kinetics of substrate inhibition by APS are quantitatively consistent with APS (B) binding to the enzyme. MgADP (EQ) complex forming a dead end EBQ complex. APS kinase rapidly loses
activity at 50C. However, a large fraction of the lost activity reappears upon incubating the heated enzyme in 0C. MgATP, MgADP, or the free nucleotides at 10 K(i) accelerate the recovery process. Indirect evidence for the action of APS kinase on APSe (the selenium analog of APS) was obtained. The rate of PAPSe formation from Se0(4)yE and MgATP by the combined action of ATP sulfurylase and APS kinase is 24% the rate of PAPS formation at the same substrate concentrations.
Impacts
(N/A)
Publications
|
Progress 01/01/82 to 12/30/82
Outputs
The sulfate activating enzumes, ATP sulfurylase and APS kinase catalyze the first two steps in the incorporation of inorganic sulfate into biological molecules. Our earlier studies on ATP sulfurylase from P. chrysogenum indicated that the enzyme had a rather low catalytic activity toward its physiological inorganic substrate, sulfate. The specific APS synthesis activity of the enzyme appeared to be about 0.13 unit x mg. protein- 1, which is only about 1% of the molybdolysis activity. The low apparent activity was not particularly troublesome because the cellular content of the enzyme was sufficient to account for the organism's growth rate on sulfate as the sole sulfur source and also, the rate at which 35(SO4)2- was converted to organic sulfur by resting cells. In the presence of excess APS kinase and inorganic pyrophosphatase, however, the pure enzyme has a specific activity of 6 to 7 units x mg. protein- 1, corresponding to an active site turnover number of at
least 400 min- 1. (The subunit molecular weight is 68,000). The apparent enhancement of ATP sulfurylase activity by APS kinase does not result from an allosteric interaction of the two enzymes (at least not one that causes an increase in the intrinsic catalytic activity of the sulfurylase).
Impacts
(N/A)
Publications
|
Progress 01/01/81 to 12/30/81
Outputs
Nitrate reductase is a cytochrome-containing molybdoflavoprotein which catalyzesthe first reaction in the assimilation of inorganic nitrate by plants and microorganisms. The fungal enzyme differs from the enzyme of higher plants in having an absolute requirement for exogenous FAD. This feature provided us with an opportunity to investigate the kinetic interactions between FAD and NADPH, and between FAD and nitrate, and between NADPH and nitrate as influenced by FAD. Our preliminary kinetics studies (supported by Hatch Funds) indicated that FAD plays an auxillary role in the NADPH-dependent reaction, perhaps serving as an electron carrier or as an activator at a site downstream from the cytochrome b component. We plan to continue our investigation of the kinetic mechanism of nitrate reductase from Penicillium chrysogenum. One goal is to clarify the role of the "second" FAD site. In our first series of new experiments, we will examine the mechanism of the reaction
using FADH(2) as the primary electron donor in place of NADPH + FAD. We have developed an anaerobic, "zero-excess dithionite" spectrophotometric assay for this purpose.
Impacts
(N/A)
Publications
|
Progress 01/01/80 to 12/30/80
Outputs
Nitrate reductase (NADPH:nitrate oxidoreductase; E.C. 1.6.6.1-3) was purified toapparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units x mg protein - 1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an S(20,w) of 7.4. The molecular weight was calculated to be 199,000. On SDS gels, the enzyme displayed two almost-contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (k(spec) ratio = 2813), FAD to FMN (k(spec) ratio = 141), and nitrate to chlorate (k(spec)ratio = 4.33), where the k(spec) (the specificity constant for a given substrate) represents V(max/K(m). The Penicillium enzyme will also catalyze the NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units x mg
protein - 1. (Km(cyt) = 1.25 x 10 - 5 M) and the reduced methyl viologen-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units x mg protein - 1 (K(mMVH)2 = 3.5 x 10 - 6 M).
Impacts
(N/A)
Publications
|
Progress 01/01/78 to 12/30/78
Outputs
ATP sulfurylase from Penicillium chrysogenum possesses eight free, reactive sulfhydryl groups (one per subunit). All eight could be modified with dithionitrobenzoic acid with no effect on enzymatic activity. Similarly, treatment of the enzyme with pyridoxal or pyridoxal phosphate (to modify lysine residues), and with 2-hydroxy-5-nitrobenzyl bromide (to modify tryptophan residues) caused no inactivation. At least 4 out of a maximum of 5 tryptophan residues/subunit were modified by the latter reagent. Treatment of the enzyme with tetranitromethane (TNM) caused a partial inhibition of activity. V(max app) was unaffected by TNM treatment but the apparent K(m) for MgATP increased 8-fold. The pseudo-first order kinetics suggest that only 1 tyrosine/subunit need be modified to account for the observed inhibition. The essential tyrosine appears to have a pK(a) less than 7.0 (lowered to about 6.0 after nitration). The enzyme could be protected from the TNM-promoted
partial inhibition by APS and MgATP plus nitrate. N-acetylimidazole, another tyrosine-specific reagent, also inactivated the enzyme. About 76% of the original activity could be restored by treating the acetylated enzyme with hydroxylamine. The cumulative results show that arginine, histidine, and tyrosine residues are essential for the functional integrity of the enzyme. However, the ability of the enzyme to bind ( 3H)ATP was not significantly affected by any of the modifications that affected catalytic activity.
Impacts
(N/A)
Publications
|
Progress 01/01/77 to 12/30/77
Outputs
Choline-O-sulfate (the sulfur acid ester of choline) is synthesized by a wide variety of fungi and plants. Choline sulfokinase, the enzyme responsible for the synthesis of the ester was purified 30-fold from mycelium of Penicillium chrysogenum, and characterized. The K(m) for PAPS is 12 muM. The enzyme is remarkably specific for the adenosine 3', 5' (or 2'-5')-diphosphate moiety. 3', 5'-ADP (PAP) has a K(i) of 2.5 to 14 and 5'-ADP are at least 300-fold higher. The enzyme is also highly specific for choline (k(m) equal to 17 MuM). Of a number of other amino alcohols tested, none were potent inhibitors and only dimethylaminoethanol served as a reasonably good substrate (K(m) equal to 800 MuM; V equal to 35% of V with choline). Trienthylaminoethanol was a significantly poorer substrate (K(m) equal to 2800 MuM; V equal to 2% of V with choline). The purified enzyme is relatively stable when stored frozen in the presence of 25% sucrose. In the absence of sucrose, the
maximum activity decreases and the K(m) for choline increases. (The K(m) for PAPS remains constant.) The age-inactivated enzyme can be restored to full activity (original V and K(m) for choline) by a 10-min preincubation with 50 mM mercaptoethanol.
Impacts
(N/A)
Publications
|
Progress 01/01/76 to 12/30/76
Outputs
Although our work is concerned with the transport and metabolism of specific sulfur and nitrogen compounds, our recent results are of use to many other researchers. For example, we have shown that membrane transport systems can be treated kinetically as multireactant ligand binding systems. Our procedures canbe used to analyze other H + symport systems and cation-dependent transport systems. The equations and diagnostic procedures that were developed should be understandable and useful to a wide variety of biochemists, microbiologists, andphysiologists who are interested in making net velocity and isotope exchange measurements of membrane transport systems. The same approach is applicable to several soluble enzymes (e.g., phosphoglucomutase). Our recent work on ATP sulfurylase provides two items of general use to workers studying enzyme kinetics. First, we call attention to the use of multiple inhibitors in probingthe basis of ordered reactions. Second, we point
out a potential pitfall in theuse of dead-end inhibitors in distinguishing between ordered and random sequences: The kinetics of dead-end inhibition are diagnostic only if the inhibitor binds to the same enzyme species as the substrate with which the inhibitor is competitive.
Impacts
(N/A)
Publications
|
Progress 01/01/75 to 12/30/75
Outputs
The regulation of inorganic sulfate transport in filamentous fungi was studied. Equations expressing the effect of internal, unlabeled sulfate on the rate of labeled sulfate transport were derived using the principles of steady state enzyme kinetics. The experimental observations of "transinhibition" agreed withthe "Iso Uni Uni" model proposed for carrier-mediated active transport. Cycloheximide, a potent inhibitor of protein synthesis, was shown to have an immediate inhibitory effect on amino acid transport in fungi. The data suggest that cycloheximide labelizes membrane-bound calcium, making the calcium available for chelation by medium constituents. A variety of lipid-soluble, weak acids were shown to inhibit membrane transport in fungi without a proportional decrease in cellular ATP. The results are in agreement with the chemiosmotic theory of active transport emergization. A kinetic analysis of theeffects of pH on transport suggest a 1:1 stoichiometry
between substrate and H(+) ion during uptake, with H(+) adding to the membrane carrier before the substrate. The choline sulfatase of Pseudomonas aeruginosa was purified and thekinetics of choline-O-sulfate hydrolysis studied. Unlike many other esterases, the choline sulfatase reaction appears to proceed via an Ordered Bi Bi mechanism.
Impacts
(N/A)
Publications
|
|