STABILITY INVESTIGATIONS ON THE PYRUVATE DECARBOXYLASE FROM ZYMOMONAS-MOBILIS

Kinetic parameters of pyruvate decarboxylase (PDC) (EC 4.1.1.1) from Zymomonas mobilis have been determined in different buffers over the range of pH 6.0-6.5. PDC revealed half-maximal saturation concentrations (K-m) of 1.1-1.3 mM pyruvate and maximal velocities (V-max) of 120-150 units/mg in Mes/KOH, potassium phosphate, imidazole and glycine-phosphate buffers. By contrast, the data obtained in sodium citrate buffer suggest a 3-fold higher affinity for the substrate pyruvate (K-m=0.45 mM), while the V-max. is 20-46% lower compared with that in the other buffer systems. PDC exhibits low stability in buffers of pH less than 5.5 and more than 8.5, while it is relatively stable in neutral and even weakly alkaline buffers, provided that the cofactors thiamin diphosphate and Mg2+ are present in sufficient amounts. Addition of sulphates such as Na2SO4 and MgSO4 stabilize PDC even in acidic buffer solutions, while chlorides are destabilizing and enhance aggregation. PDC is stable to thermal denaturation up to 60 degrees C. Thermal denaturation is irreversible and it coincides with aggregation [midpoint of the thermal-inactivation curve (T-m 63 degrees C)]. None of the tested chaotropic additives (urea, guanidium chloride, guanidine sulphate) were able to prevent aggregation. Additives like dithiothreitol and (NH4)(2)SO4 enhance stability (T-m 65.4 degrees C).