THE HYPERTHERMOPHILIC GLYCOLYTIC ENZYME ENOLASE IN THE ARCHAEON, PYROCOCCUS-FURIOUSUS - COMPARISON WITH MESOPHILIC ENOLASES

High enolase activity, as measured by the conversion of 2-phosphoglyce rate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimall y at 100 degrees C). In this organism, the enzyme probably functions i n 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% inactivat ion 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 an d yeast enzymes required a metal ion for activity, but whereas the yea st enzyme has an absolute requirement for Mg2+, the P. furiosus enolas e was equally active in the presence of Mn2+. Both enzymes were compet itively inhibited by citrate. P. furiosus enolase, as for mesophilic e nolases, probably has a homodimeric structure with subunit M(r) greate r 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 r ange of other organisms including bacteria, yeast, birds, and mammals. Substantial differences in the thermal properties of the hyperthermop hilic enzyme compared with that from less extreme thermophiles and mes ophiles might be due to a substantially enhanced composition of hydrop hobic amino acids.