In vitro Thermal and Salt Stability of Pyruvate Kinase Are Increased by Proline Analogues and Trigonelline

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The nitrogenous compounds N-methyl-L-proline (MP), trans-4-hydroxy-N-methyl-L-proline (MHP) and trigonelline (T), which undergo stress-induced accumulation in some Australian plants, were analysed and compared with proline (P) and glycinebetaine (B) for possible protective roles. The activity of pyruvate kinase (PK), prepared from Zea mays leaves and rabbit muscle, was unaffected even in the presence of 750 mM of the proline analogues. Thus, MP and MHP, like P and B, have the properties to act in vivo as compatible osmotica. T was not as compatible, decreasing enzyme activity 20% at 0.5 M. Like P and B, however, MP, MHP and T all also exhibited protective properties. They increased, in vitro, the thermal stability of PK from both plant and animal sources, and they protected PK (Zea mays) from salt inhibition at two substrate levels. The effect of salt on PK (Zea mays) was substrate dependent; at low phosphoenolpyruvate (PEP) levels, salt inhibited the enzyme activity, while salt effects were less severe in the presence of higher substrate levels. In the presence of high NaCl concentrations, the protective effects of high substrate levels and the compatible solutes seem to be additive. The Km (PEP) value of the plant PK increased in the presence of salt but the effect was ameliorated by the compatible solute MHP.