Characterization of the glycolytic enzyme enolase which is abundant in the hyperthermophilic archaeon, Pyrococcus furiosus

摘要

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolphyruvate, was found in the cytoplasm of Pyrococcus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100(degree)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 (degree)C, and a pH optimum of 8.1. The enzyme was extremely thermostable with a half time for inactivation at 100(degree)C of 40 min. In contrast, an enolase from yeast was inactivated in 1 min at 88(degree)C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg(sup ++) the P. furiosus enolase was equally active in the presence of Mn(sup ++). Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(sub 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.