Phosphoenolpyruvate Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus

A number of unique microorganisms that thrive at temperatures of 90°C or higher have been isolated in the last 2 decades. One of the best studied of these so-called hyperthermophiles is the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100°C by the fermentation of carbohydrates and peptides (15). The organism uses a modified Embden-Meyerhof glycolytic pathway that contains several novel enzymes (13, 23). For example, both hexokinase and phosphofructokinase are ADP- rather than ATP-dependent enzymes (24, 48). In addition, the expected glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase enzymes, which would convert glyceraldehyde-3-phosphate to 3-phosphoglycerate with the concomitant phosphorylation of ADP to ATP and reduction of NAD+ to NADH, appear to be replaced by a single enzyme, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR). GAPOR oxidizes glyceraldehyde-3-phosphate directly to 3-phosphoglycerate and uses ferredoxin rather than NAD+ as the electron acceptor. Purified GAPOR is a unique tungsten-containing enzyme that has no known analog in mesophilic archaea or bacteria (31). Another unusual step in glucose catabolism includes the conversion of acetyl coenzyme A (acetyl-CoA) to acetate. In anaerobic bacteria, this is a two-step process via acetyl phosphate catalyzed by phosphotransacetylase and acetate kinase. In P. furiosus, however, these two enzymes are replaced by acetyl-CoA synthetase, which converts acetyl-CoA directly to acetate and phosphorylates ADP to ATP (29). Other enzymes involved in glucose oxidation that have been purified from Pyrococcus species include pyruvate:ferredoxin oxidoreductase (4), enolase (36), and triosephosphate isomerase (25). In contrast to the enzymes described previously, these are quite similar (except in thermostability) to their mesophilic counterparts. P. furiosus also synthesizes glucose during growth on peptide-derived amino acids (15), but less work has been done to explore the gluconeogenic pathway. This is thought to occur by a reversal of the conventional Embden-Meyerhof pathway, and the activities of the enzymes proposed to be involved have been measured in cell extracts (44). Of the gluconeogenic-specific enzymes, only phosphoglycerate kinase (19) has been purified from Pyrococcus species. Herein we focus on the enzyme that carries out the first step in the conversion of pyruvate to glucose, phosphoenolpyruvate (PEP) synthetase, which catalyzes the phosphorylation of pyruvate according to equation 1: 1 Transcriptional analyses indicated that the cellular concentration of PEP synthetase increased when a glycolytic substrate such as maltose was added to the growth medium of P. furiosus (38, 39). To explain this result, it was suggested that this enzyme functions in glycolysis (reverse of equation 1) as well as in gluconeogenesis (39). However, there are no kinetic data to support this contention. It was, therefore, of some relevance to determine the catalytic properties of PEP synthetase from this organism. Surprisingly, little is known about PEP synthetases from prokaryotes, in spite of their importance in controlling carbon flow during glucose metabolism (20, 28, 37, 45, 47). The only one to be studied extensively is that from Escherichia coli (10, 11, 32, 33, 34). It is a cofactorless homodimer with a molecular mass of 150 kDa and catalyzes the phosphorylation of pyruvate by using ATP to generate PEP, AMP, and phosphate. Only two other PEP synthetases have been purified and examined to any extent, and both are from archaea, the moderately thermophilic methanogen Methanobacterium thermoautotrophicum (14) and the heterotrophic hyperthermophile Staphylothermus marinus (8, 9, 16, 17). The quaternary structure of the former enzyme was not reported, but S. marinus PEP synthetase exists as a large homomultimeric complex containing 24 subunits (17). Conversely, while the kinetic properties of the latter enzyme are unknown, those of the M. thermoautotrophicum enzyme are similar to those of E. coli PEP synthetase (14). It was therefore of some interest to investigate the physical and catalytic properties of the enzyme from P. furiosus, especially as the enzyme has been reported to be sensitive to inactivation by oxygen (44). For example, did the enzyme exist as a large complex, did it function in both gluconeogenesis and glycolysis, did it utilize nucleotides other than ATP as the phosphate donor for PEP synthesis, and what may be the cause of its oxygen sensitivity?