New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

Abstract

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent catalysis.

New views on an old enzyme: allosteric regulation and evolution of archaeal pyruvate kinases

“…Commensurate with this, SsoPK, MjaPK, MhuPK, and MmaPK were not activated by potassium (Table ). PKs from the hyperthermophilic Thermoproteales, as well as Aigarchaeota and Desulfurococcales, contain a variable amino acid (Ser, Gln, Arg, or Gly) in place of lysine and thus constitute a third group of PK enzymes . These PKs were also not stimulated by potassium (Table ).…”

“…Relatively little is known about the properties of PKs from archaea in general, but those from the hyperthermophilic crenarchaeota Thermoproteus tenax, Aeropyrum pernix, Pyrobaculum aerophilum and Thermofilum pendens have been analyzed in detail . These PKs are homotetrameric and highly thermostable enzymes that show cooperative binding to PEP.…”

“…However, in contrast to bacteria and eukarya, they do not show an allosteric response towards the classical sugar phosphate activators . These enzymes are also unusual by not requiring K + for enzyme activation . The crystal structure of PK from P. aerophilum (PaePK), a member of the hyperthermophilic Thermoproteales, has been solved and revealed an unexpected sulphate molecule (derived from the crystallization solution) bound at a site that is equivalent to the phospho‐binding site of sugar phosphates in the C domain .…”

Unusual Phosphoenolpyruvate (PEP) Synthetase-Like Protein Crucial to Enhancement of Polyhydroxyalkanoate Accumulation in Haloferax mediterranei Revealed by Dissection of PEP-Pyruvate Interconversion Mechanism

“…PYK is commonly a homotetrameric protein and is conserved in all three domains of life, with the critical function of phosphoryl transfer from PEP to ADP yielding pyruvate and ATP in all glucose-degrading organisms (17). Based on the phylogenetic analysis, PYKs can be broadly classified into K ϩ dependent and K ϩ independent, depending on the presence of conserved glutamate in the K ϩ binding site of PYKs (16,(18)(19)(20). PYKs have been identified and characterized from several Crenarchaeota, e.g., Thermoproteus tenax, Aeropyrum pernix, Pyrobaculum aerophilum, and Sulfolobus solfataricus and thermophilic Euryarchaeota, for example, T. acidophilum, Thermococcus kodakarensis, and Archaeoglobus fulgidus (18,19,(21)(22)(23).…”

An overview of structure, function, and regulation of pyruvate kinases

“…PyKs of hyperthermophilic archaea Thermoproteales (but not thermophilic bacteria) are activated by 3‐phosphoglycerate (3PG) and not by FBP, FDP, or AMP . In the glycolytic pathway of these archaea, glyceraldehyde 3‐phosphate is irreversibly oxidized to 3PG .…”