Glyceraldehyde-3-phosphate Ferredoxin Oxidoreductase, a Novel Tungsten-containing Enzyme with a Potential Glycolytic Role in the Hyperthermophilic Archaeon Pyrococcus furiosus
1995; Elsevier BV; Volume: 270; Issue: 15 Linguagem: Inglês
10.1074/jbc.270.15.8389
ISSN1083-351X
AutoresSwarnalatha Mukund, Michael W. W. Adams,
Tópico(s)Radioactive element chemistry and processing
ResumoThe archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of carbohydrates to yield acetate, CO2, and H2. Cell-free extracts contain very low activity of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, but extremely high activity of glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). GAPOR was purified under strictly anaerobic conditions. It is a monomeric, O2-sensitive protein of Mr ∼ 63,000 which contains pterin and approximately 1 tungsten and 6 iron atoms per molecule. The enzyme oxidized glyceraldehyde-3-phosphate (Km 28 μM) to 3-phosphoglycerate and reduced P. furiosus ferredoxin (Km 6 μM), but it did not oxidize formaldehyde, acetaldehyde, glyceraldehyde, benzaldehyde, glucose, glucose 6-phosphate, or glyoxylate, nor did it use NAD(P) as an electron acceptor. It is proposed that GAPOR has a glycolytic role and functions in place of glyceraldehyde-3-phosphate dehydrogenase and possibly phosphoglycerate kinase. The archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of carbohydrates to yield acetate, CO2, and H2. Cell-free extracts contain very low activity of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, but extremely high activity of glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). GAPOR was purified under strictly anaerobic conditions. It is a monomeric, O2-sensitive protein of Mr ∼ 63,000 which contains pterin and approximately 1 tungsten and 6 iron atoms per molecule. The enzyme oxidized glyceraldehyde-3-phosphate (Km 28 μM) to 3-phosphoglycerate and reduced P. furiosus ferredoxin (Km 6 μM), but it did not oxidize formaldehyde, acetaldehyde, glyceraldehyde, benzaldehyde, glucose, glucose 6-phosphate, or glyoxylate, nor did it use NAD(P) as an electron acceptor. It is proposed that GAPOR has a glycolytic role and functions in place of glyceraldehyde-3-phosphate dehydrogenase and possibly phosphoglycerate kinase. Hyperthermophiles are a recently discovered group of microorganisms that have the remarkable property of growing at temperatures of 90°C and above (1Stetter K.O. Fiala G. Huber G. Huber R. Segerer G. FEMS Microbiol. Rev. 1990; 75: 117-124Crossref Google Scholar, 2Adams M.W.W. Annu. Rev. Microbiol. 1993; 47: 627-658Crossref PubMed Scopus (299) Google Scholar). Almost all are classified as Archaea (formerly Archaebacteria, Ref. 3Woese C.R. Kandler O. Wheelis M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4576-4579Crossref PubMed Scopus (4444) Google Scholar), and the majority are strictly anaerobic heterotrophs that reduce elemental sulfur (S°) to H2S. The best studied of these is Pyrococcus furiosus (Topt 100°C), which also grows well in the absence of S° (4Fiala G. Stetter K.O. Arch. Microbiol. 1986; 145: 56-61Crossref Scopus (703) Google Scholar). This organism utilizes both complex (starch, glycogen) and simple (maltose, cellobiose) sugars as a carbon source but monosaccharides (glucose, fructose) do not support growth. The fermentation products are acetate, H2and CO2. Alanine is also produced at high partial pressures of H2(5Kengen S.W.M. Stams A.J.M. Arch. Microbiol. 1994; 161: 168-175Crossref Scopus (97) Google Scholar). The growth of P. furiosus is dependent upon tungsten (6Bryant F.O. Adams M.W.W. J. Biol. Chem. 1989; 264: 5070-5079Abstract Full Text PDF PubMed Google Scholar), an element seldom used in biological systems (7Adams M.W.W. King R.B. Encyclopedia of Inorganic Chemistry. John Wiley & Sons, New York1994: 4284-4291Google Scholar), and two tungsten-containing enzymes, aldehyde ferredoxin (Fd) 1The abbreviations used are:FdferredoxinEDEntner-DoudoroffEMEmbden-MeyerhofGAPglyceraldehyde 3-phosphateGAPDHglyceraldehyde-3-phosphate dehydrogenasePGKphosphoglycerate kinaseGAPORglyceraldehyde-3-phosphate ferredoxin oxidoreductaseAORaldehyde ferredoxin oxidoreductaseFORformaldehyde ferredoxin oxidoreductasePORpyruvate oxidoreductaseFNORferredoxin NADP oxidoreductaseEPPSN-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid. 1The abbreviations used are:FdferredoxinEDEntner-DoudoroffEMEmbden-MeyerhofGAPglyceraldehyde 3-phosphateGAPDHglyceraldehyde-3-phosphate dehydrogenasePGKphosphoglycerate kinaseGAPORglyceraldehyde-3-phosphate ferredoxin oxidoreductaseAORaldehyde ferredoxin oxidoreductaseFORformaldehyde ferredoxin oxidoreductasePORpyruvate oxidoreductaseFNORferredoxin NADP oxidoreductaseEPPSN-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid. oxidoreductase (AOR, Refs. 8Mukund S. Adams M.W.W. J. Biol. Chem. 1990; 265: 11508-11516Abstract Full Text PDF PubMed Google Scholar and 9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar) and formaldehyde Fd oxidoreductase (FOR, Refs. 10Johnson J.L. Rajagopalan K.V. Mukund S. Adams M.W.W. J. Biol. Chem. 1993; 268: 4848-4853Abstract Full Text PDF PubMed Google Scholar and 11Mukund S. Adams M.W.W. J. Biol. Chem. 1993; 268: 13592-13600Abstract Full Text PDF PubMed Google Scholar) have been purified. AOR oxidizes a range of aliphatic and aromatic aldehydes in vitro (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar), whereas FOR utilizes only C1-C3 aldehydes. FOR is thought to function in peptide catabolism (11Mukund S. Adams M.W.W. J. Biol. Chem. 1993; 268: 13592-13600Abstract Full Text PDF PubMed Google Scholar), whereas it was hypothesized that AOR catalyzed glyceraldehyde oxidation (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar) as part of an unusual nonphosphorylated Entner-Doudoroff (ED) pathway. Most of the other enzymes of this ED pathway were subsequently demonstrated in cell extracts, albeit with very low activities (12Schäfer T. Schönheit P. Arch. Microbiol. 1992; 158: 188-202Crossref Scopus (92) Google Scholar).From a very recent study, however, which used NMR and enzymatic analyses to investigate the metabolism of [13C]glucose by cell suspensions of P. furiosus, it was concluded that this occurs via an unusual Embden-Meyerhof (EM) pathway which contains ADP- (AMP-forming) rather than ATP-dependent hexose(-phosphate) kinases (13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar). A similar study by others (14Schäfer T. Xavier K.B. Santos H. Schönheit P. FEMS Microbiol. Lett. 1994; 121: 107-114Crossref Scopus (32) Google Scholar) also concluded that an EM type of pathway was present in P. furiosus, although a version of the ED pathway was also proposed. It is not clear, however, how an EM-type pathway could function in this organism, because cell extracts contain very low activities of two key glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar, 12Schäfer T. Schönheit P. Arch. Microbiol. 1992; 158: 188-202Crossref Scopus (92) Google Scholar, 13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar, 15Schäfer T. Schönheit P. Arch. Microbiol. 1993; 159: 354-363Crossref Scopus (68) Google Scholar). In fact, these enzymes are thought to function in gluconeogenesis when P. furiosus is grown on pyruvate (15Schäfer T. Schönheit P. Arch. Microbiol. 1993; 159: 354-363Crossref Scopus (68) Google Scholar). We show here that P. furiosus contains high activity of a new type of enzyme, glyceraldehyde-3-phosphate Fd oxidoreductase (GAPOR). This is proposed to function in place of GAPDH and possibly PGK in the novel EM-type pathway. Moreover, GAPOR represents the third tungsten-containing enzyme to be purified from P. furiosus.MATERIALS AND METHODSGrowth of the Organism and Enzyme PurificationP. furiosus (DSM 3638) was grown in the absence of S° at 90°C in a stainless steel 600-liter fermentor using maltose as the carbon source (6Bryant F.O. Adams M.W.W. J. Biol. Chem. 1989; 264: 5070-5079Abstract Full Text PDF PubMed Google Scholar). GAPOR was purified from 100 g (wet weight) of frozen cells under strictly anaerobic conditions at 23°C. In all chromatography steps, the columns were washed with at least 1 column volume of buffer before the absorbed proteins were eluted. The techniques and procedures were the same as those used to purify P. furiosus AOR (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar), up to and including the first chromatography step, except that sodium dithionite and glycerol were omitted from all buffers. GAPOR activity eluted from the first column (5.0 × 10 cm) of DEAE-Sepharose Fast Flow (Pharmacia Biotech Inc.) at 0.12 M NaCl in buffer A (50 m M Tris/HCl, pH 8.0, containing 2 m M dithiothreitol). Fractions containing GAPOR activity (762 mg, 6,200 units) were applied to a column (2.5 × 10 cm) of hydroxyapatite (Bio-Rad) previously equilibrated with buffer A at 2.0 ml/min. Absorbed proteins were eluted with a linear gradient (1,000 ml) from 0 to 200 m M K+ phosphate in buffer A at 2 ml/min. GAPOR activity eluted as 10 m M K+ phosphate was applied. Active fractions (310 mg, 5,250 units) were concentrated by ultrafiltration with an Amicon-type PM-30 membrane and applied to a column (1.6 × 60 cm) of Superdex 200 (Pharmacia) equilibrated with buffer A containing 200 m M KCl at 3 ml/min. GAPOR-containing fractions (50 mg, 4,250 units) were concentrated and washed with buffer A and then applied to a column (1.2 × 10 cm) of Blue Sepharose (Pharmacia) equilibrated with buffer A. Absorbed proteins were eluted with a linear gradient (120 ml) from 0 to 300 m M KCl. GAPOR activity eluted as 180 m M KCl was applied. Those fractions containing homogenous GAPOR as judged by electrophoresis (11 mg, 1,540 units) were concentrated and stored as pellets in liquid N2.Enzyme Assays and Other MethodsThe activity of GAPOR was determined at 70°C by the reduction of benzyl viologen (3.0 m M) measured at 580 nm (molar absorbance 7,800 M−1 cm−1) in 100 m M EPPS buffer, pH 8.4 (see Ref. 9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar), using glyceraldehyde 3-phosphate (GAP, 0.4 m M) as the substrate. GAP was determined to have a half-life at 70°C of 2 min under routine assay conditions (as determined using rabbit muscle GAPDH (Sigma) at 23°C). Therefore, it was added to start the reaction in all assays of GAPOR, and activities were calculated from the first 10 s of benzyl viologen reduction. The activities of AOR (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar) and FOR (11Mukund S. Adams M.W.W. J. Biol. Chem. 1993; 268: 13592-13600Abstract Full Text PDF PubMed Google Scholar) were determined at 80°C by the oxidation of crotonaldehyde and formaldehyde, respectively. GAPDH activity was measured at 70°C by following the reduction of NAD at 340 nm (16Zwickl P. Fabry S. Bogedain C. Haas A. Hensel R. J. Bacteriol. 1990; 172: 4329-4338Crossref PubMed Google Scholar). For all enzymes, 1 unit of activity is defined as 1 μmol of substrate oxidized per min. Concentrations of 3-phosphoglycerate (0-2.0 m M) were estimated using chicken liver 3-phosphoglycerate dehydrogenase (Sigma) at 23°C. Molecular weight estimation by gel filtration (Superdex 200) and SDS-electrophoresis, protein analyses, plasma emission and fluorescence spectroscopy, colorimetric iron determination, and N-terminal amino acid sequence analysis were all carried out as described previously (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar, 17Ma K. Adams M.W.W. J. Bacteriol. 1994; 176: 6509-6517Crossref PubMed Google Scholar). The pterin was extracted from purified GAPOR by I2/KI treatment (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar). NADP reduction using GAP (0.4 m M) as the substrate was determined at 70°C using a system containing GAPOR (1.5 mg/ml), P. furiosus ferredoxin (0.38 mg/ml), P. furiosus ferredoxin NADP oxidoreductase (0.1 mg/ml), and NADP (0.4 m M) under previously described conditions (18Ma K. Zhou Z.H. Adams M.W.W. FEMS Microbiol. Lett. 1994; 122: 263-266Crossref Scopus (54) Google Scholar).RESULTS AND DISCUSSIONPurification of GAPORAnaerobically prepared cytoplasmic extracts of three different batches of P. furiosus cells grown on maltose contained high GAPOR activity (4.7-8.2 units/mg with benzyl viologen as the electron acceptor, 1.2-2.6 units/mg using P. furiosus ferredoxin as the electron acceptor; see below), but the GAPDH activity was very low (0.07-0.09 unit/mg using the same cell extracts). Under aerobic conditions, cell extracts lost about 50% of GAPOR activity after 12 h at 23°C, but no activity was lost if the extract was maintained under argon. The enzyme was therefore purified under anaerobic conditions but in the absence of the O2-scavenger, sodium dithionite, which is an inhibitor (see below). Using DEAE-Sepharose Fast Flow chromatography, GAPOR and FOR activities (eluting at 0.12 M NaCl) were separated from related activities in the cell-free extract such as AOR (0.18 M NaCl) and GAPDH (0.24 M NaCl). GAPOR also co-eluted with FOR from a hydroxyapatite column but the two were easily separated by Superdex S-200 chromatography (data not shown). Two separate purification procedures each yielded about 11 mg of purified GAPOR with a specific activity of about 140 units/mg. A 30-fold purification was achieved compared with the cell-free cytoplasmic extract.Physical Properties of GAPORAs shown in Fig. 1, purified GAPOR prepared by heating at 100°C for 10 min with SDS (1.0%, w/v) gave rise to two protein bands after SDS-electrophoresis (10%, w/v, acrylamide) which corresponded to approximate Mrvalues of 44,000 and 63,000. If the sample was not heated, only the Mr44,000 band was observed (Fig. 1), suggesting that this form of the enzyme was not completely unfolded. Incomplete denaturation by SDS treatment has also been reported for some other enzymes from P. furiosus (see Refs. 2Adams M.W.W. Annu. Rev. Microbiol. 1993; 47: 627-658Crossref PubMed Scopus (299) Google Scholar and 6Bryant F.O. Adams M.W.W. J. Biol. Chem. 1989; 264: 5070-5079Abstract Full Text PDF PubMed Google Scholar). However, if purified GAPOR was precipitated with trichloroacetic acid (10%, w/v) prior to the heat treatment step, only the Mr63,000 band was evident (Fig. 1). This band was therefore assumed to represent the fully denatured protein. Both protein bands gave the same N-terminal sequence (see below), confirming that they are different denatured forms of the same protein. Analysis of GAPOR by gel filtration (in the presence of 1.0 M NaCl) gave an Mrvalue of 60,000 ± 5,000. Together with the electrophoretic data, these results suggest that the enzyme is a monomeric protein of Mrapproximately 63,000. The presence of a single subunit was confirmed by N-terminal amino acid sequence analysis of a solution of GAPOR which gave rise to a single sequence (M K F S V L K L D V G K R E V E A Q E I E R E D I F G V V D Y G I M R H N E). This showed no homology to the N-terminal sequences of either AOR or FOR from P. furiosus (11Mukund S. Adams M.W.W. J. Biol. Chem. 1993; 268: 13592-13600Abstract Full Text PDF PubMed Google Scholar) or to GAPDH from Pyrococcus woesei (16Zwickl P. Fabry S. Bogedain C. Haas A. Hensel R. J. Bacteriol. 1990; 172: 4329-4338Crossref PubMed Google Scholar).GAPOR contained 0.85 ± 0.12 tungsten and 5.0 ± 1.6 iron g atoms/mol when analyzed by plasma emission spectroscopy and 5.9 ± 0.4 iron g atoms/mol by colorimetric analysis (each value is the average of two determinations using two different enzyme preparations). The enzyme as isolated gave a broad visible absorption band around 420 nm, and this decreased by nearly 50% upon addition of sodium dithionite (data not shown), consistent with the presence of iron-sulfur chromophores. The spectra were very similar to those obtained with P. furiosus AOR, which included a broad feature near 550 nm in the oxidized form (see Fig. 1 of Ref. 8Mukund S. Adams M.W.W. J. Biol. Chem. 1990; 265: 11508-11516Abstract Full Text PDF PubMed Google Scholar). Material extracted from GAPOR after acid denaturation and oxidation with I2/KI gave fluorescence spectra that were also very similar to those obtained after treating P. furiosus AOR in a similar manner (see Fig. 1 of Ref. 9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar). These spectra are characteristic of a pterin-type cofactor, suggesting that the tungsten in GAPOR is present as a tungstopterin center, as it is in AOR and also FOR (10Johnson J.L. Rajagopalan K.V. Mukund S. Adams M.W.W. J. Biol. Chem. 1993; 268: 4848-4853Abstract Full Text PDF PubMed Google Scholar). The N-terminal sequence of GAPOR showed no homology to the subunits of the two other types of tungsten-containing enzymes that are known, carboxylic acid reductase (19White H. Feicht R. Huber C. Lottspeich F. Simon H. Biol. Chem. Hoppe-Seyler. 1991; 372: 999-1005Crossref PubMed Scopus (53) Google Scholar) and formylmethanofuran dehydrogenase (20Schmitz R.A. Richter M. Linder D. Thauer R.K. Eur. J. Biochem. 1992; 209: 1013-1018Crossref PubMed Scopus (62) Google Scholar). Notably, the tungstoenzymes AOR, FOR, and carboxylic acid reductase share a high degree of N-terminal homology (11Mukund S. Adams M.W.W. J. Biol. Chem. 1993; 268: 13592-13600Abstract Full Text PDF PubMed Google Scholar).Catalytic Properties of GAPORGAPOR was purified by its ability to oxidize GAP at 70°C using benzyl viologen as the electron acceptor. Since GAP had a half-life under assay conditions of 2 min, it was important to establish that a degradation product of GAP was not serving as a substrate for GAPOR. This was substantiated by the following. When GAP was completely degraded by a 5-min incubation at 80°C under assay conditions (there was no reduction of benzyl viologen during this process); upon the addition of GAPOR, the reduction of benzyl viologen was observed only when freshly prepared GAP was subsequently added. These results also indicated that the thermal degradation products of GAP did not inhibit GAPOR. Using GAP at concentrations of 0.01-0.50 m M (benzyl viologen, 3 m M), the Km and Vmvalues obtained from a linear double-reciprocal plot were 30 μM and 350 units/mg, respectively. There was significant inhibition of GAPOR using GAP concentrations above 0.5 m M. Using benzyl viologen at concentrations of 0.01-5.0 m M (GAP, 0.40 m M), the Km and Vmvalues were 0.43 m M and 310 units/mg, respectively. To investigate whether 3-phosphoglycerate was the product of GAPOR activity, the reaction was allowed to proceed under standard assay conditions at 70°C for 2 min. The reaction mixture was then rapidly cooled to 4°C, and the concentration of 3-phosphoglycerate was determined enzymatically to be 95 μM. This compares with a calculated value of 110 μM, based on the initial rate of benzyl viologen reduction. 3-Phosphoglycerate could not be detected if the same experiment was performed in the absence of GAPOR, which also shows that 3-phosphoglycerate was not a significant thermal degradation product of GAP.No activity was detected in the routine assay of GAPOR when benzyl viologen was replaced by NADP or NAD (each 5 m M), but P. furiosus ferredoxin did function as an electron carrier for the enzyme. The Km and Vmvalues calculated from a linear Lineweaver-Burk plot were approximately 6 μM and 90 units/mg, respectively (using 0.1-50 μM ferredoxin and 0.40 m M GAP). Using GAP at concentrations of 0.01-0.50 m M (ferredoxin, 100 μM), the Km and Vmvalues obtained were 28 μM and 78 units/mg, respectively. The high activity obtained with ferredoxin as the electron carrier and the high affinity of GAPOR for both GAP and ferredoxin suggest that these are the physiological substrates. Since NADPH rather than reduced ferredoxin is the physiological electron donor to the H2-evolving hydrogenase of P. furiosus (18Ma K. Zhou Z.H. Adams M.W.W. FEMS Microbiol. Lett. 1994; 122: 263-266Crossref Scopus (54) Google Scholar), it was of interest to determine if GAP oxidation could be coupled to NADP reduction. A combination of GAPOR, ferredoxin, and P. furiosus ferredoxin NADP oxidoreductase (FNOR, Ref. 17Ma K. Adams M.W.W. J. Bacteriol. 1994; 176: 6509-6517Crossref PubMed Google Scholar) reduced NADP at a rate of 88 μmol/min/mg (of FNOR) at 70°C, suggesting that in vivo, reductant from the GAPOR reaction could be readily converted to NADPH and disposed of via hydrogenase as H2.There was no detectable reduction of benzyl viologen when GAP was replaced in the routine assay of GAPOR at 70°C by acetaldehyde, crotonaldehyde, formaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, phenylacetaldehyde, glyceraldehyde (measured at 40°C, see Ref. 9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar), glucose, glucose 6-phosphate, dihydroxyacetone phosphate, glycerol 3-phosphate, or glyoxalate, each at a concentration of 1.0 or 10 m M. Similarly, there was no significant effect on the activity of GAPOR using GAP as the substrate when glucose, glucose 6-phosphate, dihydroxyacetone phosphate, glycerol 3-phosphate, or phosphoglycolate, each at a concentration of 10 m M, were included in the routine assay. The presence of crotonaldehyde (10 m M) or formaldehyde (10 m M) decreased GAPOR activity by approximately 25%, whereas acetyl phosphate, 3-phosphoglycerate, or 2,3-bisphosphoglycerate (each 10 m M) stimulated GAPOR activity by between 58 and 93%.When sodium dithionite was added to GAPOR (1.0 mg/ml in 50 m M Tris/HCl, pH 8.0) to a final concentration of 3.0 m M, less than 50% of the GAP oxidation activity was observed. When the dithionite was then removed from the enzyme by gel filtration, approximately 70% of the original specific activity was recovered. The substrate specificity of GAPOR was unaffected when the enzyme was prepared with and without dithionite. The substrate specificities of AOR and FOR from P. furiosus (which are purified in the presence of sodium dithionite) were also unaffected when sodium dithionite was removed (in particular, GAP was not a substrate for either enzyme). GAPOR was routinely assayed in the absence of phosphate ions and its activity was unaffected by the presence of 5 m M K+ phosphate in the assay medium. However, K+ phosphate at a concentration of 200 m M stimulated GAPOR activity by approximately 3.5-fold, and Na+ arsenate (200 m M) had a similar effect. By analogy with GAPDH, which generates 1,3-bisphosphoglycerate from GAP, these results suggest that GAPOR in the presence of phosphate might also produce 1,3-bisphosphoglycerate. We could not detect 1,3-bisphosphoglycerate (using rabbit muscle GAPDH at 25°C) as a product of the GAPOR reaction when the standard assay was carried out for 2 min at 70°C in the presence of 200 m M K+ phosphate, whereas the concentration of 3-phosphoglycerate produced under these conditions was determined enzymatically to be 285 μM, which was in good agreement with the calculated value (350 μM, based on the initial rate of benzyl viologen reduction). This result is not conclusive, however, because 1,3-bisphosphoglycerate is very unstable and rapidly hydrolyzes to 3-phosphoglycerate and inorganic phosphate (even at 25°C, Ref. 21Negelin E. Methods Enzymol. 1957; 3: 216-220Crossref Scopus (21) Google Scholar), and so it may have rapidly degraded under assay conditions. On the other hand, GAPOR activity was also stimulated about 3.5-fold by the presence of potassium chloride, sodium citrate, or sodium sulfate (each 200 m M), showing that there is general enhancement of GAPOR activity by high ionic strength and particularly by polyanions, including phosphate.Physiological Function of GAPORThe oxidation of GAP to 1,3-bisphosphoglycerate and its conversion to 3-phosphoglycerate is usually accomplished by a combination of the glycolytic enzymes, GAPDH and PGK. The activities of these two enzymes in maltose-grown P. furiosus are both very low when assayed in the glycolytic direction (≤0.06 units/mg at 90°C, Refs. 13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar and 15Schäfer T. Schönheit P. Arch. Microbiol. 1993; 159: 354-363Crossref Scopus (68) Google Scholar), yet it has been shown that an EM-type glycolytic pathway is the predominant route for glucose oxidation in this organism (13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar). It therefore seems reasonable to suggest that GAPOR is a glycolytic enzyme and functions in place of GAPDH and PGK. As shown in Fig. 2, it is proposed that GAPOR produces 3-phosphoglycerate and that the reductant is disposed of as H2via NADPH (see Ref. 18Ma K. Zhou Z.H. Adams M.W.W. FEMS Microbiol. Lett. 1994; 122: 263-266Crossref Scopus (54) Google Scholar). An attractive feature of this pathway is that all of the enzymes involved exhibit-specific activities of greater than 0.5 units/mg at 70−90°C using cell-free extracts of maltose-grown P. furiosus (2Adams M.W.W. Annu. Rev. Microbiol. 1993; 47: 627-658Crossref PubMed Scopus (299) Google Scholar, 6Bryant F.O. Adams M.W.W. J. Biol. Chem. 1989; 264: 5070-5079Abstract Full Text PDF PubMed Google Scholar, 12Schäfer T. Schönheit P. Arch. Microbiol. 1992; 158: 188-202Crossref Scopus (92) Google Scholar, 13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar, 15Schäfer T. Schönheit P. Arch. Microbiol. 1993; 159: 354-363Crossref Scopus (68) Google Scholar, 17Ma K. Adams M.W.W. J. Bacteriol. 1994; 176: 6509-6517Crossref PubMed Google Scholar). Moreover, assuming that the ADP required for hexose(-phosphate) activation is readily available from anabolic processes (13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar), the proposed pathway yields 4 mol of ATP/mol of glucose oxidized to acetate, which is in accordance with growth yield data (see Refs. 12Schäfer T. Schönheit P. Arch. Microbiol. 1992; 158: 188-202Crossref Scopus (92) Google Scholar, 13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar, and 22Kengen S.W.M. Stams A.J.M. FEMS Microbiol. Lett. 1994; 117: 305-310Crossref Scopus (35) Google Scholar). We cannot exclude the possibility that in vivo GAPOR produces 1,3-bisphosphoglycerate, but this would seem unlikely since (a) if 1,3-bisphosphoglycerate was used by PGK to generate ATP, the overall ATP yield (6 ATP/glucose) would exceed that determined experimentally (see Refs. 12Schäfer T. Schönheit P. Arch. Microbiol. 1992; 158: 188-202Crossref Scopus (92) Google Scholar and 22Kengen S.W.M. Stams A.J.M. FEMS Microbiol. Lett. 1994; 117: 305-310Crossref Scopus (35) Google Scholar), and (b) the activities of both PGK and GAPDH in P. furiosus increase dramatically (up to 10-fold) in pyruvate-grown cells, suggesting they function primarily in gluconeogenesis (15Schäfer T. Schönheit P. Arch. Microbiol. 1993; 159: 354-363Crossref Scopus (68) Google Scholar).FIG. 2Proposed role of GAPOR in the conversion of glucose to acetate in P. furiosus. Enzymes that have been purified are shown in bold and underlined, unusual enzymes are indicated, and dotted lines indicate electron transfer reactions. Data were taken from Refs. 2Adams M.W.W. Annu. Rev. Microbiol. 1993; 47: 627-658Crossref PubMed Scopus (299) Google Scholar, 13Kengen S.W.M. Debok F.A.M. Vanloo N.D. Dijkema C. Stams A.J.M. Devos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar, 17Ma K. Adams M.W.W. J. Bacteriol. 1994; 176: 6509-6517Crossref PubMed Google Scholar, and 18Ma K. Zhou Z.H. Adams M.W.W. FEMS Microbiol. Lett. 1994; 122: 263-266Crossref Scopus (54) Google Scholar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The EM-type pathway shown in Fig. 2 also satisfies the requirement of ATP production during glucose oxidation to pyruvate (22Kengen S.W.M. Stams A.J.M. FEMS Microbiol. Lett. 1994; 117: 305-310Crossref Scopus (35) Google Scholar), which does not occur in the ED-type pathway proposed for P. furiosus (12Schäfer T. Schönheit P. Arch. Microbiol. 1992; 158: 188-202Crossref Scopus (92) Google Scholar). Indeed, the operation of this ED pathway is also questionable in light of the very low activities of some of the enzymes involved (12Schäfer T. Schönheit P. Arch. Microbiol. 1992; 158: 188-202Crossref Scopus (92) Google Scholar). A notable exception in this regard is AOR (9Mukund S. Adams M.W.W. J. Biol. Chem. 1991; 266: 14208-14216Abstract Full Text PDF PubMed Google Scholar), which perhaps necessitates a reevaluation of its physiological function. In any event, we have demonstrated that P. furiosus GAPOR is distinct from AOR and also from FOR, the other tungstoenzymes in this organism. GAPOR is also unrelated to GAPDH or to any other known tungstoprotein. The enzyme is present at a relatively high cellular concentration (about 3% of the total protein), consistent both with its function in the primary pathway of sugar metabolism and with the dependence of cell growth upo
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