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• W2008156512 abstract "ATP sulfurylase from Penicillium chrysogenum is an allosteric enzyme in which Cys-509 is critical for maintaining the R state. Cys-509 is located in a C-terminal domain that is 42% identical to the conserved core of adenosine 5′-phosphosulfate (adenylylsulfate) (APS) kinase. This domain is believed to provide the binding site for the allosteric effector, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Replacement of Cys-509 with either Tyr or Ser destabilizes the R state, resulting in an enzyme that is intrinsically cooperative at pH 8 in the absence of PAPS. The kinetics of C509Y resemble those of the wild type enzyme in which Cys-509 has been covalently modified. The kinetics of C509S resemble those of the wild type enzyme in the presence of PAPS. It is likely that the negative charge on the Cys-509 side chain helps to stabilize the R state. Treatment of the enzyme with a low level of trypsin results in cleavage at Lys-527, a residue that lies in a region analogous to a PAPS motif-containing mobile loop of true APS kinase. Both mutant enzymes were cleaved more rapidly than the wild type enzyme, suggesting that movement of the mobile loop occurs during the R to T transition. ATP sulfurylase from Penicillium chrysogenum is an allosteric enzyme in which Cys-509 is critical for maintaining the R state. Cys-509 is located in a C-terminal domain that is 42% identical to the conserved core of adenosine 5′-phosphosulfate (adenylylsulfate) (APS) kinase. This domain is believed to provide the binding site for the allosteric effector, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Replacement of Cys-509 with either Tyr or Ser destabilizes the R state, resulting in an enzyme that is intrinsically cooperative at pH 8 in the absence of PAPS. The kinetics of C509Y resemble those of the wild type enzyme in which Cys-509 has been covalently modified. The kinetics of C509S resemble those of the wild type enzyme in the presence of PAPS. It is likely that the negative charge on the Cys-509 side chain helps to stabilize the R state. Treatment of the enzyme with a low level of trypsin results in cleavage at Lys-527, a residue that lies in a region analogous to a PAPS motif-containing mobile loop of true APS kinase. Both mutant enzymes were cleaved more rapidly than the wild type enzyme, suggesting that movement of the mobile loop occurs during the R to T transition. adenosine 5′-phosphosulfate (adenylylsulfate) 3′-phosphoadenosine 5′-phosphosulfate (3′-phosphoadenylylsulfate) Magnesium chelate of ATP (“MgATP” solutions contained the indicated concentration of total ATP plus a 5 mm excess of MgCl2 thereby maintaining a constant fraction of the total ATP in the MgATP form (approximately 90% at pH 8.0) as the nucleotide concentration was varied (30Wood H.G. Davis J.J. Lochmüller H. J. Biol. Chem. 1966; 241: 5692-5704Abstract Full Text PDF PubMed Google Scholar, 31Storer A.C. Cornish-Bowden A. Biochem. J. 1976; 159: 1-5Crossref PubMed Scopus (241) Google Scholar)) N-ethylmaleimide 5′,5′-dithiobis(2-nitrobenzoate) 2-(N-morpholino)ethanesulfonic acid polymerase chain reaction Hill coefficient free enzyme “relaxed” and “taut” structural states of allosteric enzymes ATP sulfurylase (MgATP:SO42−adenylyltransferase, EC 2.7.7.4) catalyzes the first intracellular reaction in the incorporation of inorganic sulfate into organic molecules by sulfate assimilating organisms:MgATP+SO42−⇌MgPPi+APSAPS1 is then phosphorylated to PAPS in a reaction catalyzed by the second sulfate-activating enzyme, APS kinase, (MgATP:adenosine 5′-phosphosulfate 3′ phosphotransferase EC 2.7.1.25):MgATP+APS⇌PAPS+MgADPATP sulfurylase from the filamentous fungus Penicillium chrysogenum is an oligomer composed of six identical 64-kDa subunits (573 residues). Each subunit possesses three free SH (cysteinyl) groups, 2Among fungal ATP sulfurylases that have been examined so far, two Cys residues (Cys-42 and Cys-509) are conserved. The third one in P. chrysogenum (Cys-68) replaces a Val that is present at that position in other fungal ATP sulfurylases. of which only one (designated SH-1) can be modified by sulfhydryl-reactive reagents such as DTNB and NEM under nondenaturing conditions (1Renosto F. Schultz T. Re E. Mazer J. Chandler C.J. Barron A. Segel I.H. J. Bacteriol. 1985; 164: 674-683Crossref PubMed Google Scholar). Complete modification of SH-1 (six per hexamer) changes the initial velocity kinetics at pH 8 from normal-hyperbolic (Hill coefficient,n H = 1) to sigmoidal (n Happroximately 2) with a concomitant increase in the [S]0.5 values for MgATP and SO42− (or MoO42−);V max app at a fixed subsaturating cosubstrate level is reduced (2Renosto F. Martin R.L. Segel I.H. J. Biol. Chem. 1987; 262: 16279-16288Abstract Full Text PDF PubMed Google Scholar). A number of experimental approaches, including protection against chemical inactivation by reversibly bound ligands (2Renosto F. Martin R.L. Segel I.H. J. Biol. Chem. 1987; 262: 16279-16288Abstract Full Text PDF PubMed Google Scholar), direct binding measurements (3Martin R.L. Daley L.A. Lovric Z. Wailes L.M. Renosto F. Segel I.H. J. Biol. Chem. 1989; 264: 11768-11775Abstract Full Text PDF PubMed Google Scholar), and single turnover isotope trapping (3Martin R.L. Daley L.A. Lovric Z. Wailes L.M. Renosto F. Segel I.H. J. Biol. Chem. 1989; 264: 11768-11775Abstract Full Text PDF PubMed Google Scholar), established that the sigmoidal curves reflected true cooperative binding as opposed to a kinetically based phenomenon. The dramatic effect of in vitro modification of SH-1 suggested several possible scenarios, including that modification induces a conformational state in the enzyme that is normally inducedin vivo by a reversibly bound allosteric effector. The effector was subsequently shown to be PAPS (4Renosto F. Martin R.L. Wailes L.M. Daley L.A. Segel I.H. J. Biol. Chem. 1990; 265: 10300-10308Abstract Full Text PDF PubMed Google Scholar). Further experiments established that the enzyme from several other fungi behaved identically to the P. chrysogenum enzyme, whereas ATP sulfurylases from rat liver (5Yu M. Martin R.L. Jain S. Chen L.J. Segel I.H. Arch. Biochem. Biophys. 1989; 269: 156-174Crossref PubMed Scopus (37) Google Scholar), spinach leaf (6Renosto F. Patel H.C. Martin R.L. Thomassian C. Zimmerman G. Segel I.H. Arch. Biochem. Biophys. 1993; 307: 272-285Crossref PubMed Scopus (76) Google Scholar), cabbage leaf (7Osslund T. Chandler C. Segel I.H. Plant Physiol. 1982; 70: 39-45Crossref PubMed Google Scholar), yeast (4Renosto F. Martin R.L. Wailes L.M. Daley L.A. Segel I.H. J. Biol. Chem. 1990; 265: 10300-10308Abstract Full Text PDF PubMed Google Scholar), and the Riftia bacterial symbiont (8Renosto F. Martin R.L. Borrell J.L. Nelson D.C. Segel I.H. Arch. Biochem. Biophys. 1991; 290: 66-78Crossref PubMed Scopus (41) Google Scholar) didnot respond in the same way to Cys modification or to PAPS. The cumulative results indicated that (a) fungal ATP sulfurylase possesses an allosteric PAPS binding site that is not present in the enzyme from other sources and (b) SH-1 is either in the region of, or in communication with, the PAPS binding site. Fungal sulfurylase was subsequently shown to possess a C-terminal region (approximately residues 396–539) that is 42% identical to the conserved core of APS kinase (9Foster B.A. Thomas S.M. Mahr J.A. Renosto F. Patel H. Segel I.H. J. Biol. Chem. 1994; 269: 19777-19786Abstract Full Text PDF PubMed Google Scholar, 10MacRae I. Rose A.B. Segel I.H. J. Biol. Chem. 1998; 273: 28583-28589Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 11MacRae I.J. Segel I.H. Fisher A.J. Biochemistry. 2000; 39: 1613-1621Crossref PubMed Scopus (44) Google Scholar), a protein with a high affinity for PAPS. SH-1 is Cys-509, which is located in the APS kinase-like C-terminal domain, a few residues upstream from a putative PAPS motif (12Satishchandran C. Hickman Y.N. Markham G.D. Biochemistry. 1992; 31: 11684-11688Crossref PubMed Scopus (46) Google Scholar). It is likely that residues 396–540 of P. chrysogenum ATP sulfurylase evolved from true APS kinase and that this region provides the allosteric binding site for PAPS. In effect, the C-terminal region of fungal ATP sulfurylase is a regulatory subunit that happens to be covalently linked to the catalytic subunit. 3For a while, the GenBank™ entry for P. chrysogenum ATP sulfurylase (accession number A53651) described the enzyme as a “probable PAPS synthetase” and suggested that the enzyme possesses APS kinase activity. This information (which was not submitted by us) is incorrect and contrary to published accounts. The homogeneous enzyme does not have measurable APS kinase activity (<0.001 units × mg of protein−1 under standard assay conditions where true APS kinase from P. chrysogenumexhibits about 25 units × mg of protein−1). Yeast ATP sulfurylase (GenBank™ accession number S55198) was described in similar terms. But this enzyme is even less likely to possess APS kinase activity, considering that it does not possess an APS kinase-like region. In contrast, the enzyme from Aquifex aeolicus (GenBank™ accession number AE000722), which also possesses a C-terminal APS kinase-like domain, may very well be bifunctional, considering the similarity of the P-loop and PAPS motif sequences to those of true APS kinases. Our preliminary hypothesis (in terms of the concerted transition model) was that covalent modification of Cys-509 promotes the same R to T allosteric transition (13Monod J. Wyman J. Changeux J.-P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6150) Google Scholar, 14Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience, New York1993: 421-464Google Scholar) as does PAPS binding. The inhibition of P. chrysogenum ATP sulfurylase by PAPS may be the way that fungi prevent PAPS accumulation to toxic levels. Another consideration is that in fungi, PAPS is a major branch point metabolite of sulfate assimilation. One branch leads to cysteine and other reduced sulfur compounds; the other branch to choline-O-sulfate, a sulfur storage compound and/or osmoprotectant (15Ballio A. Chain E.B. Dentice di Accadia F. Navizio F. Rossi C. Ventura M.T. Sel. Sci. Papers Istituto Superiore Sanita. 1959; 2: 343-353Google Scholar, 16Itahashi M. J. Biochem. (Tokyo). 1961; 50: 52-61Crossref PubMed Scopus (9) Google Scholar, 17Renosto F. Segel I.H. Arch. Biochem. Biophys. 1977; 180: 416-428Crossref PubMed Scopus (14) Google Scholar, 18Hanson A.D. Rathinasabapathi B. Chamberlin B. Gage D.A. Plant Physiol. 1991; 97: 1199-1205Crossref PubMed Scopus (88) Google Scholar). Thus the inhibition may be part of a more extensive sequential feedback process. In contrast, yeasts and most bacteria do not form large quantities of sulfate esters, whereas plants (and some bacteria) preferentially use APS (rather than PAPS) as the substrate for the reductive assimilation of sulfate. In other words, PAPS is not at a branch point in these other organisms. The objective of the present study was to establish the role of Cys-509 in stabilizing the R state. To this end, we investigated the kinetic consequences of replacing Cys-509 with either tyrosine or serine. Mutations in codon 509 were made by PCR amplification of the C-terminal 221 base pairs of the fungal ATP sulfurylase gene (codons 506–573). This sequence begins with an indigenous XhoI site 3 base pairs upstream from codon 509 and ends after the stop codon with an engineered XbaI site. Each PCR used a cloned cDNA copy of the native gene as the template, the C-terminal coding primer PcATS308 (5′-GGTCTAGATCTTACTGACGCTCCAGGAAACCC-3′), and an upstream primer containing the XhoI site and the desired mutation. Upstream primers with their respective produced mutations were as follows: PcATS315 (C059S), 5′-TCCCCTCGAGCACTCTGAGCAGTCCG-3′; PcATS317 (C509Y), 5′-TCCCCTCGAGCACTACGAGCAGTCCG-3′. All PCRs were carried out using the DNA polymerase Pfu (Stratagene). The resulting 221-base pair DNAs were subcloned as XhoI-XbaI fragments into a pBluescript KS(+) plasmid containing a cDNA clone of fungal ATP sulfurylase in which the wild type C-terminal 221 base pairs had been removed. All cloned PCR fragments were sequenced to ensure that the desired mutations were introduced. Sequenced ATP sulfurylase genes were cloned asNdeI-BglII fragments into the Novagen pET23a(+) plasmid and introduced into Escherichia coli strain BL21(DE3) for protein expression. About 0.2 ml of an 8-h culture was used to inoculate two 3-liter Fernbach flasks each containing 1000 ml of LB ampicillin medium. The cultures were grown aerobically at 37 °C for 8–10 h and then transferred to 15 °C. Upon transfer to 15 °C, 1 g of α-lactose was added per liter of culture to induce protein expression. After 8–10 h at 15 °C, the cells were harvested by centrifugation at 12,000 ×g for 10 min. Approximately 4–8 ml of packed cells was obtained. The cells were then resuspended in about 50 ml of chilled 40 mm Tris-Cl, pH 8.0, and lysed in a single pass through a Watts Fluidair Microfluidizer (model B12–04DJC M3). All subsequent steps were carried out at 4 °C. Cell debris and unbroken cells were removed by centrifuging at 16,000 × g for 10 min. The supernatant fluid was applied to a blue dextran (19Ryan L.D. Vestling C.S. Arch. Biochem. Biophys. 1974; 160: 279-284Crossref PubMed Scopus (229) Google Scholar) column (2.5 × 10 cm) that had been equilibrated with 40 mm Tris-Cl, pH 8.0. The column was then washed with the same buffer at 6 ml/min until the effluent had an A 280 nm of 0.005 or less. Protein was eluted with a linear gradient of NaCl (0–0.7m) in 40 mm Tris-Cl, pH 8.0 (total volume 500 ml) at a flow rate of 2 ml per min. 7-ml fractions were collected, and their A 280 nm and ATP sulfurylase activity were measured. Fractions containing enzyme activity (coincident with the major protein peak) were pooled (total volume approximately 85 ml), dialyzed against 40 mm Tris-Cl, pH 8.0, and then applied to a DEAE-cellulose column (2.5 × 10 cm) equilibrated in the same buffer. After a brief wash, protein was eluted at 1 ml per min with a linear gradient of NaCl (0–0.4 m) in 40 mmTris-Cl, pH 8.0 (total volume, 400 ml). Seven fractions containing ATP sulfurylase activity (total volume 49 ml) were pooled, divided into 1-ml aliquots, and stored frozen. A typical preparation yielded about 25 mg of pure enzyme. TheA 280 nm/A 260 nm ratio of the enzymes ranged from 1.91 (for C509Y) to 2.01 (for C509S). SDS gel electrophoresis indicated that all the enzymes were at least 95% pure. The absence of Cys-509 in the mutant enzymes was confirmed by demonstrating their lack of reactivity with DTNB in the absence of SDS (1Renosto F. Schultz T. Re E. Mazer J. Chandler C.J. Barron A. Segel I.H. J. Bacteriol. 1985; 164: 674-683Crossref PubMed Google Scholar). Most biochemicals, buffers, column media, and coupling enzymes were obtained from Sigma. PAPS was prepared as described previously (20MacRae I. Segel I.H. Arch. Biochem. Biophys. 1997; 337: 17-26Crossref PubMed Scopus (16) Google Scholar). Concentrations of stock solutions were established by enzymatic analysis using Nuclease P1 coupled to ATP sulfurylase, hexokinase, and glucose-6-phosphate dehydrogenase in the presence of excess PPi, MgCl2, NADP+, and 1 mm glucose. ATP sulfurylase concentrations were determined from the relationship: concmg × ml−1 =A 280 nm/0.871 (21Tweedie J.W. Segel I.H. Prep. Biochem. 1971; 1: 91-117Crossref PubMed Scopus (21) Google Scholar). (In theory, this results in a 3% error in the assumed concentration of C509Y.) ATP sulfurylase activity was characterized by the continuous, coupled spectrophotometric molybdolysis assay (22Segel I.H. Renosto F. Seubert P.A. Methods Enzymol. 1987; 143: 334-349Crossref PubMed Scopus (51) Google Scholar) in the presence of NADH, P-enolpyruvate, KCl, excess adenylate kinase, inorganic pyrophosphatase, sulfate-free pyruvate kinase + lactate dehydrogenase, and approximately 0.5 μg (0.02 unit) of pure P. chrysogenum APS kinase (10MacRae I. Rose A.B. Segel I.H. J. Biol. Chem. 1998; 273: 28583-28589Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 22Segel I.H. Renosto F. Seubert P.A. Methods Enzymol. 1987; 143: 334-349Crossref PubMed Scopus (51) Google Scholar, 23Renosto F. Seubert P.A. Segel I.H. J. Biol. Chem. 1984; 259: 2113-2123Abstract Full Text PDF PubMed Google Scholar). The stoichiometry of the assay is 2 mol of NADH oxidized per mol of AMP formed. In addition to providing good sensitivity, this assay has the advantage in that both primary substrates, MgATP and MoO42−, are continuously regenerated. The APS kinase serves to remove traces of APS formed from contaminating inorganic sulfate during the preincubation period (20MacRae I. Segel I.H. Arch. Biochem. Biophys. 1997; 337: 17-26Crossref PubMed Scopus (16) Google Scholar). (APS is a potent product inhibitor of the enzyme, whereas the small increment of PAPS formed is innocuous.) Unless indicated otherwise, all assays were conducted at 30 °C, in 50 mm Tris-Cl, pH 8.0. The total MgCl2 present was always 5 mm greater than that of the total ATP. The specific activities of the wild type, C509S, and C509Y forms of the enzyme freshly purified from the E. coli expression system and assayed at 5 mm total ATP, 10 mm total Mg2+ (as MgCl2), and 10 mmMoO42− were, in order, 20, 17, and 14.5 units × mg of protein−1. 1 unit is the amount of enzyme that catalyzes the formation of 1 μmol of primary product in 1 min. For each experimental velocity curve, theV max value and the Hill coefficient,n H, were determined by fitting the plottedv versus [substrate] data to the Hill equation: v=Vmax[S] nHK′+[S] nHEquation 1 Hill coefficients were also determined as the slope of the Hill plot,logvVmax−v=nHlog[S]−logK′,Equation 2 in the region corresponding to 50% saturation (i.e.where log [v/(V max −v)] = 0) or over the range corresponding to 10–90% saturation (14Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience, New York1993: 421-464Google Scholar). Curve-fits were obtained using DeltaGraph 4.05c (Macintosh) with all points weighted equally. Then H of a single plot determined by the three methods generally agreed to within 0.1. The n Hof replicate curves obtained at different times generally agreed to within <0.15. Although the Hill coefficient was useful for comparing the sigmoidicity of different velocity curves, ultimately, differences in n H need to be related to the complete velocity equation for an allosteric bireactant enzyme (see “”). Fig.1 shows the velocity curves of the C509Y mutant enzyme under standard assay conditions. The most striking feature of the curves is that they are sigmoidal in the absence of PAPS. In fact, the increase in n H with increasing concentrations of the fixed cosubstrate is the same trend displayed by the wild type enzyme after covalent modification of Cys-509 (data not shown). 4The native enzyme modified with NEM at Cys-509 yielded the following data: The n H of thev versus [MgATP] plots increased from 1.2 at 0.2 mm MoO42− to 2.0 at 5 mm MoO42−. Then H of the v versus[MoO42−] plots increased from 1.8 at 0.3 mm MgATP to 2.1 at 5 mm MgATP. Up to this point, the results suggested that cooperative behavior is induced by either (a) increasing the bulk of the side chain at position 509 or (b) eliminating the negative charge (R-S−) at this position. It was thought that replacing Cys-509 with the slightly smaller and uncharged Ser might help to distinguish between these two possibilities. Fig.2 shows the velocity curves of the C509S enzyme at several different fixed concentrations of cosubstrate. Despite the size similarity of Ser and Cys, the plots are again sigmoidal, although, compared with C509Y, C509S has a lower [S]0.5 for either substrate at any given concentration of cosubstrate. Also, unlike the curves shown in Fig. 1, then H values of the v versus[MgATP] plots for C509S do not change significantly with increasing [MoO42−]. At subsaturating MgATP, thev versus [MoO42−] curves are also sigmoidal, but n H approaches unity as the concentration of MgATP approaches saturation. This trend is consistent with the preferential binding of MgATP to free E of the R state. That is, as the fixed [MgATP] approaches saturation, the enzyme is driven far toward the R state, which binds MoO42−in a normal hyperbolic manner. At 5 mm MgATP, the K m for MoO42−is 0.1 mm, which is the same as that of the noncooperative wild type enzyme. In terms of Equation 9 (), L app for C509S at saturating MgATP (equivalent to Lc) must be very small, implying that c is less than unity. The sigmoidicity of thev versus MoO42−plot at subsaturating MgATP can be attributed, at least in part, to the synergism between MgATP and MoO42−. That is, even if K ibT =K ibR (e = 1), andK mbT = K mbR(j = 1), the v versus[B] plots can be sigmoidal at subsaturating [A] if (a) substrate A binds preferentially to the R state (c < 1), and (b) the substrates bind to the R state synergistically (f < 1). The last condition seems highly likely given that the R state should closely resemble the noncooperative wild type enzyme where theK m value for each substrate is smaller than the corresponding K i value (1Renosto F. Schultz T. Re E. Mazer J. Chandler C.J. Barron A. Segel I.H. J. Bacteriol. 1985; 164: 674-683Crossref PubMed Google Scholar). The bireactant kinetics of C509S are similar to those of the wild type enzyme in the presence of PAPS. 5The wild type enzyme yielded the following data at 50 μm PAPS: The nH of the v versus [MgATP] plots varied from approximately 1.5 at 0.1 mm MoO42− to 1.7 at 1 mmMoO42−. The nH of the v veresus [MoO4] plots decreased from approximately 2 to 0.05 mm MgATP to approximately 1 at 2.5 mm MgATP. Comparing the above results with those of the C509Y enzyme leads to the conclusions that either (a) substituting a Tyr residue for Cys-509 drives the enzyme much further toward the T state than does substituting a Ser at this position or (b) the T state induced by substituting Tyr at position 509 is structurally different from that induced by substituting Ser (see “Discussion”). In either case, the results show that cooperative behavior is a not simply a result of increasing the bulk of the residue at position 509. Either the negative charge on the side chain of Cys-509 plays a critical role in stabilizing the R state, or the side chain size is extremely important and any change will favor a shift to the T state. Activation by a competitive inhibitor at low 6Exactly how “low” the competitive substrate must be to demonstrate activation is best established by trial and error. For a simple unireactant system where S and I bind exclusively to the R state, the peak velocity occurs at θ = n L(n−1) − α − 1, where θ = [I]/K iand α = [S]/K s. Thus as the fixed [S] is increased, the peak moves closer to the vertical axis and eventually disappears.competitive substrate concentrations is a hallmark of true cooperative binding. As shown in Fig. 3, inorganic thiosulfate, an inhibitor competitive with SO42− or MoO42− (24Seubert P.A. Hoang L. Renosto F. Segel I.H. Arch. Biochem. Biophys. 1983; 225: 679-691Crossref PubMed Scopus (30) Google Scholar), does exactly that. Activation by S2O32− is also seen with the wild type enzyme after chemical modification of Cys-509 (2Renosto F. Martin R.L. Segel I.H. J. Biol. Chem. 1987; 262: 16279-16288Abstract Full Text PDF PubMed Google Scholar), or in the presence of PAPS (4Renosto F. Martin R.L. Wailes L.M. Daley L.A. Segel I.H. J. Biol. Chem. 1990; 265: 10300-10308Abstract Full Text PDF PubMed Google Scholar, 20MacRae I. Segel I.H. Arch. Biochem. Biophys. 1997; 337: 17-26Crossref PubMed Scopus (16) Google Scholar). Note that the experimental level of the noncompetitive cosubstrate (MgATP) influences the effect of the competitive inhibitor. That is, the activation is eliminated by an MgATP concentration that is too low in the case of C509Y, or too high in the case of C509S. These opposite effects are consistent with the different effects of MgATP binding on the cooperativity of the two mutant enzymes as illustrated in Figs.1 b and 2 b. The side chain of a Ser residue is not much smaller than that of a Cys residue, but unlike Ser, a substantial fraction of the Cys side chain may be ionized at the standard assay pH of 8.0. The observation that C509S is intrinsically cooperative raised the possibility that the charge on residue 509 plays a major role in stabilizing the R state. If the side chain of Cys-509 behaves normally (i.e. has a pK a of 8.0–8.5), decreasing the assay pH from 8.0 to (e.g.) 6.5 would decrease the fraction of the residue in the Cys-S− form significantly. It was of interest then to determine whether protonating the Cys anion of the wild type enzyme had the same effect as substituting Ser for Cys. As shown in Table I, decreasing the pH did indeed induce sigmoidal v versus[MoO42−] curves. Lowering the pH also decreased V max,app and increased the [S]0.5. However, the wild type enzyme at pH 6.5 did not mimic C509S: First, the velocity curve remained sigmoidal at 5 mm MgATP (n H = 2.1). Second, the enzyme at pH 6.5 was activated by S2O32− only at high concentrations of MgATP (data not shown). In these respects, the enzyme behaved like C509Y rather than C509S. Surprisingly, then H of C509S also increased as the assay pH was decreased (n H was 1.8 at pH 8.0 and 2.3 at pH 6.5). Consequently, we cannot conclude that the sigmoidicity induced in the wild type enzyme was solely a response to protonating Cys-509. Considering the pH range studied, it is likely that protonating one or more His residues can contribute to an R to T transition. Several His residues are located in the C-terminal domain, including one adjacent to Cys-509 (His-508). His has been shown to be essential for ATP sulfurylase activity (5Yu M. Martin R.L. Jain S. Chen L.J. Segel I.H. Arch. Biochem. Biophys. 1989; 269: 156-174Crossref PubMed Scopus (37) Google Scholar, 9Foster B.A. Thomas S.M. Mahr J.A. Renosto F. Patel H. Segel I.H. J. Biol. Chem. 1994; 269: 19777-19786Abstract Full Text PDF PubMed Google Scholar, 25Venkatachalam K.V. Fuda H. Koonin E.V. Strott C.A. J. Biol. Chem. 1999; 274: 2601-2604Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 26Deyrup A.T. Singh B. Krishnan S. Lyle S. Schwartz N.B. J. Biol. Chem. 1999; 274: 28929-28936Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), a role that may account in part for the decrease in V max,app as the pH was decreased. (A decrease in the fraction of the total ATP in the MgATP form may also have contributed to the decrease inV max,app and increase in [S]0.5 as the pH was decreased.)Table IEffect of pH on some kinetic properties of wild type P. chrysogenum ATP sulfurylasepHn H[MoO42−]0.5V max appmmμmol × min −1 × mg of protein 18.01.060.3217.27.51.160.4216.37.01.660.4814.26.52.300.7212.8Rates were measured at 0.25 mm MgATP as described under “Materials and Methods,” except that the assay mixtures were buffered at the indicated pH value. The buffers were prepared by mixing 0.05 m MES, “free acid” with 0.05 m Tris “free base” to the desired pH. Although the curve fit of the pH 8 data to the Hill equation yielded an n H of 1.06 theR 2 value of the fit to the Henri-Michaelis-Menten equation (n H = 1.00) was not significantly poorer. Lower pH values could not be studied because a protein precipitate would form in the assay mixtures. Open table in a new tab Rates were measured at 0.25 mm MgATP as described under “Materials and Methods,” except that the assay mixtures were buffered at the indicated pH value. The buffers were prepared by mixing 0.05 m MES, “free acid” with 0.05 m Tris “free base” to the desired pH. Although the curve fit of the pH 8 data to the Hill equation yielded an n H of 1.06 theR 2 value of the fit to the Henri-Michaelis-Menten equation (n H = 1.00) was not significantly poorer. Lower pH values could not be studied because a protein precipitate would form in the assay mixtures. The experiments described in Table I were conducted in MES-Tris buffers in which the MES concentration increased as the pH was decreased. However, MES per se was not responsible for the sigmoidicity as evidenced by the hyperbolic velocity curves obtained in 0.05M MES (plus Tris to pH 8). It was of interest to determine whether PAPS had an additional effect on a mutant enzyme, or whether the mutation transformed the enzyme completely to the T state. As shown in Fig. 4, the n Hvalue of C509S increased further as the concentration of PAPS was increased. At 240 μm [PAPS], then H of the v versus [MgATP] plot was nearly 3. Thus the Cys to Ser mutation promoted only a partial shift toward the T state allowing the R to T equilibrium to be driven further toward the T state or back toward the R state by the appropriate ligand. In this respect, C509S resembles a typical allosteric enzyme. The apparent n H limit of 3 (instead of 6) is very likely a consequence of the nonexclusive binding of PAPS and/or substrates. However, the possibility that the enzyme behaves in an alternating “half-of-the-sites” manner cannot be immediately discarded. The effect of PAPS onV max,app indicates that either (a) the catalytic activity of the T state is much less than that of the R state, or (b) substrate binding to the T state is not highly synergistic, or (c) both conditions apply. In contrast to the results shown in Fig. 4, PAPS decreased the sigmoidicity of the v versus [MgATP] plot of C509Y: At 1 mm MoO42−in the absence of PAPS, n H andV max,app were, respectively, 2.3 and 12.2 units × mg of protein−1. At 240 μm PAPS, n H was 2.0;V max,app decreased to 6.8 units × mg of protein−1 (data not shown). As shown in Fig. 5, tre" @default.
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