Activity and Stability of Recombinant Bifunctional Rearranged and Monofunctional Domains of ATP-Sulfurylase and Adenosine 5′-Phosphosulfate Kinase
1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês
10.1074/jbc.274.16.10751
ISSN1083-351X
AutoresAndrea T. Deyrup, Srinivasan Krishnan, Bhawani Singh, Nancy B. Schwartz,
Tópico(s)Folate and B Vitamins Research
ResumoMurine adenosine 3′-phosphate 5′-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5′-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42−) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which the order of the two active domains was reversed or altered relative to the linker region. The critical role of the linker region was established by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 °C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t 1/2 of 10 min at 37 °C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast, no significant deactivation of any truncated kinase-containing recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only. Murine adenosine 3′-phosphate 5′-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5′-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42−) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which the order of the two active domains was reversed or altered relative to the linker region. The critical role of the linker region was established by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 °C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t 1/2 of 10 min at 37 °C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast, no significant deactivation of any truncated kinase-containing recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only. The formation of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) 1The abbreviations used are: PAPS, adenosine 3′-phosphate 5′- phosphosulfate; APS, adenosine 5′- phosphosulfate; IMAC, Tris-imidazole buffer; NTA, nitrilotriacetic acid.1The abbreviations used are: PAPS, adenosine 3′-phosphate 5′- phosphosulfate; APS, adenosine 5′- phosphosulfate; IMAC, Tris-imidazole buffer; NTA, nitrilotriacetic acid. (1Sugahara K. Schwartz N.B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6615-6618Crossref PubMed Scopus (106) Google Scholar), the sole donor of activated sulfate in mammalian systems, requires the sequential actions of two enzymes: ATP-sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) (see Reaction 1). ATP+SO42−→APS+PPiAPS+ATP→ADP+PAPSReaction 1 We have studied the PAPS-synthesizing enzymes in the context of a defect in the production of PAPS in the brachymorphic mutant mouse where a severe reduction in the kinase activity and a partial reduction in the sulfurylase activity was demonstrated (1Sugahara K. Schwartz N.B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6615-6618Crossref PubMed Scopus (106) Google Scholar, 2Sugahara K. Schwartz N.B. Arch. Biochem. Biophys. 1982; 214: 601-609Crossref Scopus (65) Google Scholar, 3Sugahara K. Schwartz N.B. Arch. Biochem. Biophys. 1982; 214: 589-601Crossref PubMed Scopus (67) Google Scholar). To understand this intriguing double enzyme defect, the relationship between the two activities was investigated by determining whether they represented two separate polypeptides or a single bifunctional polypeptide. The two sulfate-activating enzymes, ATP-sulfurylase and APS kinase, were purified from rat chondrosarcoma and shown to have nearly identical molecular properties and fractionation behavior (4Geller D. Henry J.G. Belch J. Schwartz N.B. J. Biol. Chem. 1987; 262: 7374-7382Abstract Full Text PDF PubMed Google Scholar). To distinguish whether a single polypeptide with multiple active sites or two tightly complexed polypeptides pertain, characterization of both kinetic mechanisms (5Lyle S. Geller D.H. Ng K. Stanczak J. Westley J. Schwartz N.B. Biochem. J. 1994; 301: 355-359Crossref PubMed Scopus (20) Google Scholar, 6Lyle S. Geller D.H. Ng K. Westley J. Schwartz N.B. Biochem. J. 1994; 301: 349-354Crossref PubMed Scopus (17) Google Scholar), as well as affinity purification of the sulfate activation complex were accomplished (7Lyle S. Stanczak J. Ng K. Schwartz N.B. Biochemistry. 1994; 33: 5920-5925Crossref PubMed Scopus (62) Google Scholar). These studies identified the mammalian sulfurylase/kinase as a bifunctional enzyme (7Lyle S. Stanczak J. Ng K. Schwartz N.B. Biochemistry. 1994; 33: 5920-5925Crossref PubMed Scopus (62) Google Scholar) that uses a channeling mechanism to transfer the intermediate APS efficiently from the sulfurylase to the kinase active site (8Lyle S. Ozeran J.D. Stanczak J. Westley J. Schwartz N.B. Biochemistry. 1994; 33: 6822-6827Crossref PubMed Scopus (40) Google Scholar, 9Lyle S. Stanczak J. Westley J. Schwartz N.B. Biochemistry. 1995; 34: 940-945Crossref PubMed Scopus (26) Google Scholar). Subsequently, the murine and human sulfurylase/kinase have been cloned, sequenced, and expressed as a fused bifunctional enzyme (10Li H. Deyrup A. Mensch Jr., J.R. Domowicz M. Konstantinidis A. Schwartz N.B. J. Biol. Chem. 1995; 270: 29453-29459Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 11.Deyrup, A. T., Structural and Functional Analysis of Mammalian ATP Sulfurylase/APS KinasePh.D. thesis, 1997, University of Chicago, Chicago, IL.Google Scholar). The finding of multiple functions on a single polypeptide suggests that this complex enzyme is a critical locus for regulation and a vulnerable site for mutations.The expressed 624-amino acid murine ATP-sulfurylase/APS kinase has an amino-terminal region (residues 1–199) that is highly homologous to known APS kinases and a carboxyl-terminal region (residues 237–624) that is similar to known ATP-sulfurylases (10Li H. Deyrup A. Mensch Jr., J.R. Domowicz M. Konstantinidis A. Schwartz N.B. J. Biol. Chem. 1995; 270: 29453-29459Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). These two domains are joined by a 37-amino acid intervening sequence (linker region) (10Li H. Deyrup A. Mensch Jr., J.R. Domowicz M. Konstantinidis A. Schwartz N.B. J. Biol. Chem. 1995; 270: 29453-29459Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). This arrangement in which the kinase domain is positioned at the NH2 terminus of the bifunctional enzyme whereas the sulfurylase domain is toward the COOH terminus is maintained in the human (11.Deyrup, A. T., Structural and Functional Analysis of Mammalian ATP Sulfurylase/APS KinasePh.D. thesis, 1997, University of Chicago, Chicago, IL.Google Scholar), spoonworm (12Rosenthal E. Leustek T. Gene. 1995; 165: 243-248Crossref PubMed Scopus (50) Google Scholar), and Drosophila (13Julien D. Crozatier M. Kas E. Mech. Dev. 1997; 68: 179-186Crossref PubMed Scopus (24) Google Scholar) sulfurylase/kinase, and is the reverse of the physiological reaction sequence, the gene order in the Escherichia coli operon (14Leyh T.S. Vogt T.F. Suo Y. J. Biol. Chem. 1992; 267: 10405-10410Abstract Full Text PDF PubMed Google Scholar), and the structural order of the protein product in Penicillium chrysogenumwhere the sulfurylase has a partial kinase-like sequence fused at the COOH terminus (15Foster B.A. Thomas S.M. Mahr J.A. Renosto F. Patel H.C. Segel I.H. J. Biol. Chem. 1994; 269: 19777-19786Abstract Full Text PDF PubMed Google Scholar).The number of examples of single polypeptides that carry out multiple functions is increasing, and it has been suggested that such multifunctional enzymes have selective advantages over separate monofunctional counterparts. In some cases, benefits of bifunctionality such as improved regulation (16Kirschner K. Lane A.N. Strasser A.W. Biochemistry. 1991; 30: 472-478Crossref PubMed Scopus (73) Google Scholar, 17Brzovic P.S. Ngo K. Dunn M.F. Biochemistry. 1992; 31: 3831-3839Crossref PubMed Scopus (97) Google Scholar) and enhanced stability (18Yablonski M. Pasek D. Han B. Jones M. Traut T. J. Biol. Chem. 1996; 271: 10704-10708Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) have been demonstrated. A fused sulfurylase/kinase that uses a kinetically channeled mechanism may offer advantages in overcoming obstacles inherent to the sulfate activation pathway. For instance, APS is both spontaneously labile, resulting in nonenzymatic degradation to AMP and free sulfate, and subject to breakdown by cytosolic sulfohydrolase/sulfatase activities if released into the medium (19Stokes A.M. Denner W.H. Rose F.A. Dodgson K.S. Biochim . Biophys. Acta. 1973; 302: 64-72Crossref PubMed Scopus (13) Google Scholar). The unusually high equilibrium constant (>108) for the reverse sulfurylase reaction (toward ATP formation) implies that even in the presence of associated pyrophosphatases, APS must be phosphorylated to PAPS (which is not a substrate for ATP-sulfurylase), or it will be redirected in the reverse sulfurylase reaction. Thus a channeling bifunctional protein may shift the equilibrium of the reversible sulfurylase reaction in a favorable way or protect the APS intermediate from degradative effects of the solvent.The availability of cloned and expressed sulfurylase/kinase allows us to address the question of the relationship of the bifunctional sulfurylase/kinase domains to monofunctional ATP-sulfurylases and ATP kinases and whether there is a distinct mechanistic or structural advantage to a fused protein. To examine the ability of the kinase and sulfurylase domains to function independently (as occurs in plants, fungi, and bacteria), we have generated and expressed a series of constructs that encode independent monofunctional domains. The importance of the domain order and organization was examined through the construction of several mutants in which the order of the two active domains was reversed or altered in relation to each other or to the linker region. The critical role of the linker region was then established by construction of recombinants in which the linker region was deleted or rearranged in relation to the two active domains.EXPERIMENTAL PROCEDURESRestriction enzymes were obtained from New England Biolabs, unless otherwise indicated. T4 polynucleotide kinase was from Promega, Amplitaq was from Perkin Elmer-Cetus, DNA polymerase and associated reagents for automated sequencing were from Applied Biosystems, nickel-nitrilotriacetic acid alkaline phosphatase conjugate used for Western blot analysis was from Qiagen. Pfu DNA polymerase and cloning vector pBluescript were obtained from Stratagene. The pCR 2.1 vector for direct ligation of polymerase chain reaction products was a component of the Invitrogen TA cloning kit. Thrombin protease for removal of the histidine tag, the pET-15b bacterial expression vector and metal chelate resin for gravity purification of the expressed protein were purchased from Novagen. All enzymes were used with the buffers recommended by the suppliers. Determination of protein concentration was done using the Bio-Rad protein assay and the Pierce BCA protein assay systems.Polymerase Chain Reaction and SequencingPolymerase chain reactions were performed in a Perkin Elmer GeneAmp 2400 thermal cycler using either Taq polymerase from Perkin Elmer orPfu polymerase from Stratagene. Standard cycling parameters included 1–5 min of preincubation at 94 °C followed by twenty cycles of 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C. Following the cycling step, a 10-min extension step at 72 °C allowed completion of all unfinished transcripts. All oligonucleotides were purchased from the University of Chicago oligonucleotide core facility. Automated DNA sequencing was performed on an ABI PRISM 377 DNA sequencer.Expression and Purification of Separate Enzyme DomainsThree constructs corresponding to the APS kinase region were designed: APS kinase alone (1MAPSK) (amino acid residues 1–199); APS kinase plus linker (APSKL) (residues 1–236); and finally, APS kinase plus linker and an extension into the ATP-sulfurylase region (APSK456) (residues 1–456). The latter primer (N9) was intended to introduce a TAA stop codon following Gly-456 of the sulfurylase region, but in fact altered the reading frame so that translation ran on into the downstream vector sequence, adding a 24-residue heterologous peptide sequence (NSRIRLLTKPERKLSWLLPPLSNN) to the COOH terminus of the APSK456 construct. A fourth APS kinase expression construct (APSK257) arose spontaneously during the creation of a site-directed mutant; deletion of a single nucleotide at residue 257 caused a frameshift resulting in substitution of a proline for an alanine followed by a termination codon. In addition, three ATP-sulfurylase constructs were designed corresponding to the ATP-sulfurylase domain alone (ATPS) (residues 237–624); ATP-sulfurylase plus linker (LATPS) (residues 200–624); and ATP-sulfurylase with an NH2-terminal extension into the kinase region (3MSK) (residues 70–624).Plasmid pET-15bSK1 containing the coding region for the sulfurylase/kinase was used as a polymerase chain reaction template for generation of inserts. Primers were synthesized that contained restriction enzyme cutting sites to facilitate post-amplification ligation of products into the pET-15b expression vector. The sites chosen, NdeI and XhoI, do not occur in the sulfurylase/kinase coding sequence. The amino acids Gly-Ser-His were added to the amino terminus of all constructs by vector sequences, and an additional artificial Met was added at the amino end of all constructs except 1MAPSK, APSKL, APSK456, and 3MSK.The Novagen pET-15b vector was used for bacterial expression of the separate enzyme domains. This plasmid provides elements needed for translation in E. coli as well as His-Tag and thrombin digestion site coding sequences to facilitate purification of the expressed proteins. Amplified fragments were doubly digested withNdeI and XhoI and ligated into the likewise digested vector. Recovered cloned inserts were sequenced in their entirety before transformation into JM109 DE3 cells by the CaCl2 method.Protein expression and purification were accomplished as described previously (16Kirschner K. Lane A.N. Strasser A.W. Biochemistry. 1991; 30: 472-478Crossref PubMed Scopus (73) Google Scholar). Briefly, isopropyl-1-thio-β-d-galactopyranoside was added to overnight bacterial cultures and incubation was continued for 3–4 h at 37 °C with shaking. Cells were pelleted and sonicated in IMAC 5 buffer (5 mm imidazole, 50 mm Tris, pH 7.9). Following removal of cellular debris by ultracentrifugation, the soluble protein fraction was diluted 1:2 in IMAC 5 sonication buffer and loaded onto a His-bind column from Novagen according to the supplier's protocol. Following incubation at 4 °C for 20 min with intermittent mixing, the flowthrough was collected, and the column was washed with 30 mm imidazole in 50 mm Tris, pH 7.9, to remove the majority of nonspecifically bound bacterial proteins, after which the expressed protein was eluted with 400 mm imidazole in 50 mm Tris, pH 7.9. The purified protein was dialyzed into phosphate buffer (25 mmNaH2PO4/K2HPO4, pH 7.8, 1 mm dithiothreitol, 1 mm EDTA) overnight in preparation for enzymatic assays.Western Blot AnalysisA nickel nitrilotriacetic acid (Ni-NTA) alkaline phosphatase conjugate was used to detect the His-tagged expressed protein on Western blots. Briefly, the purified protein was electrophoresed in a 12% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked in 3% bovine serum albumin in TBS (150 mm NaCl, 10 mm Tris-HCl, pH 7.4) buffer overnight and then washed three times in TBST (500 mm NaCl, 0.05% Tween 20, 20 mm Tris-HCl, pH 7.5) buffer. The blot was then incubated with the Ni-NTA alkaline phosphatase conjugate at a 1:500 dilution in TBST buffer for 2 h and washed as before. The protein band was visualized using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate in a Tris buffer (100 mm NaCl, 5 mm MgCl2, 100 mm Tris-HCl, pH 9.5) followed by a 10% trichloroacetic acid wash and storage in H2O.Generation of Rearranged Mutant ProteinsBoth the ATPS/APSK and ATPS/APSK/L constructs were created from the normal S/K cDNA clone using the methodology of Ali and Steinkasserer (21Ali S.A. Steinkasserer A. BioTechniques. 1995; 18: 746-750PubMed Google Scholar). For the former, the APS kinase region was amplified with a 5′ primer (SW1), which had no restriction site, and a 3′ primer (N5), which included aXhoI site, whereas the ATP-sulfurylase region was amplified with a 5′ primer with an NdeI site (N6) but a 3′ primer without a restriction site using Vent DNA polymerase. Following gel purification of the two bands, the blunt-ended DNA fragments were phosphorylated for 30 min at 37 °C and then ligated for 15 min at room temperature in a 10-μl reaction. 1 μl of the ligation reaction was then re-amplified using the former internal primers (N5 and N6). An additional amplification step was required in the construction of the ATPS/APSK/L mutant. The ATPS/L/APSK construct was generated through polymerase chain reaction incorporation of restriction sites 5′ and 3′ to each domain (APSK, ATPS, and linker) followed by digestion and ligation of the respective half-sites to give the desired order. AnEcoRI site was inserted between the sulfurylase and linker sequence, causing insertion of the dipeptide Glu-Phe. Similarly, aXbaI site was introduced between the linker and kinase, inserting the dipeptide Ser-Arg.Enzyme AssaysThe optimal protein amount for assessing enzymatic activity was found to be between 0.25 and 1.0 μg. Following gravity column purification, the protein concentration was determined using the Pierce BCA protein assay, and the preparation was diluted with phosphate buffer to a stock concentration of 20 μg/ml for use in all three assays. All assays were completed a minimum of three times; a representative single data set is presented.The ATP-sulfurylase assay is performed in the reverse of the physiological direction of ATP formation as described (18Yablonski M. Pasek D. Han B. Jones M. Traut T. J. Biol. Chem. 1996; 271: 10704-10708Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) because of the unfavorable K eq of the forward reaction. Standard sulfurylase assays contained 50 mmNaH2PO4/K2HPO4 (pH 7.8), 12 mm MgCl2, 0.5 mmdithiothreitol, 5 mm NaF, 0.2 mmNa4P2O7 (containing 6.7 mCi of32P), 0.1 mm APS, and 50 μl of enzyme preparation in a total volume of 1.0 ml.The standard kinase assay contained 80 nm[35S]APS, 0.5 mm ATP (pH 7.0), 5 mm MgCl2, 10 mm ammonium sulfate, and 13 μl of enzyme and was brought up to 25 μl with buffer A (25 mm NaH2PO4/K2HPO4, pH 7.8, 1 mm dithiothreitol, 1 mm EDTA, and 10% glycerol) (6Lyle S. Geller D.H. Ng K. Westley J. Schwartz N.B. Biochem. J. 1994; 301: 349-354Crossref PubMed Scopus (17) Google Scholar). Conversion of the labeled APS substrate to PAPS is monitored by paper electrophoresis, which separates the two charged species.Paper electrophoresis is also employed in the standard coupled assay, which tests the overall reaction with ATP and sulfate as substrates, measuring the yields of APS and PAPS. The standard overall reaction (25 μl) contained 0.4 mm[35S]H2SO4, 10 mmATP, 20 mm MgCl2, 22 mm Tris-HCl (pH 8.0), and 15 μl of enzyme preparation (6Lyle S. Geller D.H. Ng K. Westley J. Schwartz N.B. Biochem. J. 1994; 301: 349-354Crossref PubMed Scopus (17) Google Scholar). This assay is valuable not only as a measure of the ability of the enzyme to synthesize PAPS from ATP and SO42−, but also as an assay of the forward sulfurylase reaction.Kinetic parameters were obtained as described (5Lyle S. Geller D.H. Ng K. Stanczak J. Westley J. Schwartz N.B. Biochem. J. 1994; 301: 355-359Crossref PubMed Scopus (20) Google Scholar, 6Lyle S. Geller D.H. Ng K. Westley J. Schwartz N.B. Biochem. J. 1994; 301: 349-354Crossref PubMed Scopus (17) Google Scholar). For the forward sulfurylase, K mATP andK msulfate were found to be 350 and 37 μm, respectively, for 1MSK and 147 and 77 μm, respectively, for LATPS. Data from experiments in the physiologically reverse direction yieldedK mAPS andK mpyrophosphate of 2.3 and 32 μm, respectively, for 1MSK and 138 and 12, respectively, for LATPS. For APS-kinase, KmAPS and KmATP were found to be 88 nm and 6.2 μm, respectively, for 1MSK and 960 nm and 41 μm, respectively, for APSKL.Thermal Stability ExperimentsDetermination of the stability of the various recombinant forms of enzyme was performed by assaying sulfurylase and kinase activity after incubation of 100-μl aliquots of the various enzymes at 25 or 37 °C for various lengths of time up to a maximum incubation period of 2 h. Activities were monitored by assay procedures as described.DISCUSSIONThe ability of individual domains of some multifunctional enzymes to function independently has recently received increasing attention (18Yablonski M. Pasek D. Han B. Jones M. Traut T. J. Biol. Chem. 1996; 271: 10704-10708Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 22Beaucamp N. Hofmann A. Kellerer B. Jaenicke R. Protein Sci. 1997; 6: 2159-2165Crossref PubMed Scopus (45) Google Scholar). In the case of murine ATP-sulfurylase/APS kinase we have shown that the native bifunctional enzyme, which possesses both sulfurylase and kinase activities, efficiently converts all APS synthesized into PAPS (7Lyle S. Stanczak J. Ng K. Schwartz N.B. Biochemistry. 1994; 33: 5920-5925Crossref PubMed Scopus (62) Google Scholar, 10Li H. Deyrup A. Mensch Jr., J.R. Domowicz M. Konstantinidis A. Schwartz N.B. J. Biol. Chem. 1995; 270: 29453-29459Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The native bifunctional enzyme has an NH2-terminal region homologous to known APS kinases and a COOH-terminal region highly homologous to known ATP-sulfurylases (10Li H. Deyrup A. Mensch Jr., J.R. Domowicz M. Konstantinidis A. Schwartz N.B. J. Biol. Chem. 1995; 270: 29453-29459Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). To understand the selective advantages of the bifunctional enzyme over expressed monofunctional domains, we engineered segments of sulfurylase/kinase to produce the two individual enzyme activities on separately expressed polypeptides.The ability of the ATP-sulfurylase domain alone to catalyze the sulfurylase reaction in both the forward and reverse directions was the first evidence that the bifunctional enzyme can be divided into active fragments containing each enzyme activity. Although a construct encoding the APS kinase domain-only (1MAPSK) was inactive for kinase activity, addition of the 37-residue linker region to the COOH-terminal end of the kinase polypeptide (APSKL) restored kinase activity. However, the importance of this sequence to kinase activity cannot be attributed a priori to a catalytic or structural role, because it is possible that the APSK polypeptide is incapable of proper folding and is therefore inactive. These results do however indicate that fusion of the two activities does not preclude their ability to function independently. Furthermore, isolation of the two enzyme activities on individual polypeptides and the ability of each domain to function separately implies that each domain must have the independent ability to bind ATP and APS.Because we could construct monofunctional units that express each activity, it was of interest to determine whether they could act in a coupled reaction to synthesize PAPS, when mixed in solution. Assays of such mixtures showed that although 1MAPSK and ATPS together had no appreciable kinase or overall activity, a mixture of APSKL and ATPS displayed kinase activity and overall synthesis of PAPS comparable to those of the intact enzyme (Fig. 3). In these mixing experiments, the amount of PAPS synthesized increased when the amount of one enzyme (APSKL or ATPS) was held constant, and the other (ATPS or APSKL) was increased. Thus it appears that the APS intermediate is efficiently transferred between the two separate polypeptides. Future experiments using isotope dilution and enrichment will examine the ability of such mixtures to exhibit either efficient coupling like the separate fungal enzymes or channeling as demonstrated by the bifunctional rat chondrosarcoma enzyme (8Lyle S. Ozeran J.D. Stanczak J. Westley J. Schwartz N.B. Biochemistry. 1994; 33: 6822-6827Crossref PubMed Scopus (40) Google Scholar).Preliminary kinetic data suggest that there are modest differences in binding of the substrates, ATP and free sulfate and the product, pyrophosphate, to the sulfurylase portion of the enzyme in 1MSK and the ATPS mutant, whereas the binding of APS is significantly different. Tighter binding of APS in the native enzyme, 1MSK, may be a reflection of the native enzyme to not release the APS intermediate into the medium and instead transfer it directly to the active site of the kinase portion of the enzyme. But because APS is the product that is released from the individual ATPS enzyme, its binding efficiency is lower. There is also a significant difference in the binding of the APS intermediate to the kinase portion of 1MSK compared with the monofunctional APSKL. These data would suggest that a covalent linkage between the two domains leads to a significant perturbation in apparent binding affinities without much effect on catalytic efficiency. Detailed kinetic analyses are underway to investigate these mechanistic aspects.The results obtained from the rearranged structural mutants, ATPS/L/APSK, ATPS/APSK, APSK/ATPS, and ATPS/APSK/L are particularly interesting. For instance, the inability of the first three of these constructs to catalyze the sulfurylase reaction is difficult to interpret, especially considering the fact that the individual sulfurylase domain is capable of functioning independently, and that the fourth rearranged mutant, ATPS/APSK/L, exhibited normal sulfurylase activity comparable to that of the wild type. These results will become more comprehensible when the results of the stability experiments are considered.The inability of the APS kinase domain to function without a linker region at its COOH terminus correlates with the inability of the ATPS/APSK and ATPS/L/APSK mutants to form PAPS (<10% of the wild type enzyme). These results imply either that the linker region is needed for kinase activity in addition to its positional specificity,i.e. the linker must be present at the COOH-terminal end of the kinase domain, or that the alteration of the sequence disrupts the kinase activity caused by improper folding. The later argument can be discounted based on the result of the third construct, APSK/ATPS. This construct, although not demonstrating normal kinase activity, did show about 25% of the kinase activity of the wild type. The partial preservation of kinase activity in this recombinant may be caused by the extension of the COOH-terminal region of the kinase domain by additional sulfurylase amino acid sequence. Interestingly when the linker sequence is aligned with the ATPS sequence (Fig.4) there is a region toward the NH2 terminus of the ATPS sequence (residues 290–318 in the ATPS sequence) that is similar to the linker (23.3% identity in 30-amino acid overlap) and may be responsible for the part
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