The hyperthermophilic glycolytic enzyme enolase in the archaeon, Pyrococcus furiosus: comparison with mesophilic enolases.

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100 degrees C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of > 90 degrees C and a pH optimum of 8.1. The enzyme was extremely thermostable with a time for 50% inactivation at 100 degrees C of 40 min. In contrast, an enolase from yeast was totally inactivated in 1 min at 88 degrees C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg2+, the P. furiosus enolase was equally active in the presence of Mn2+. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(r) greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals. Substantial differences in the thermal properties of the hyperthermophilic enzyme compared with that from less extreme thermophiles and mesophiles might be due to a substantially enhanced composition of hydrophobic amino acids.