Comparative Analysis of Pyruvate Kinases from the Hyperthermophilic Archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the Hyperthermophilic Bacterium Thermotoga maritima

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50kDa subunits. The enzymes required divalent cations, Mg and Mn being most effective, but were independent of K . Temperature optima for activity were 85 °C (A. fulgidus) and above 98 °C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 °C (A. pernix) and showed melting temperatures for thermal unfolding at 93 °C (A. fulgidus) or above 98 °C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K ions, had a temperature optimum of 80 °C, was highly thermostable up to 90 °C, and had a melting temperature above 98 °C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.