STABILITY INVESTIGATIONS ON THE PYRUVATE DECARBOXYLASE FROM ZYMOMONAS-MOBILIS

Kinetic parameters of pyruvate decarboxylase (PDC) (EC 4.1.1.1) from Z ymomonas mobilis have been determined in different buffers over the ra nge 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 120-15 0 units/mg in Mes/KOH, potassium phosphate, imidazole and glycine-phos phate 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 e ven weakly alkaline buffers, provided that the cofactors thiamin dipho sphate and Mg2+ are present in sufficient amounts. Addition of sulphat es such as Na2SO4 and MgSO4 stabilize PDC even in acidic buffer soluti ons, while chlorides are destabilizing and enhance aggregation. PDC is stable to thermal denaturation up to 60 degrees C. Thermal denaturati on is irreversible and it coincides with aggregation [midpoint of the thermal-inactivation curve (T-m 63 degrees C)]. None of the tested cha otropic 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).