Abstract
SUMMARY: The pyruvate carboxylase of Pseudonomas fluorescens was purified 160-fold from cells grown on glucose at 20 °. The activity of this purified enzyme was not affected by acetyl-coenzyme A or L-aspartate, but was strongly inhibited by ADP, which was competitive towards ATP. Pyruvate gave a broken double reciprocal plot, from which two apparent Km values could be determined, namely 0.08 and 0.21 mM, from the lower and the higher concentration ranges, respectively. The apparent Km for HCO-3 at pH 6.9, in the presence of the manganese ATP ion (MnATP2-), was 3.1 mM. The enzyme reaction had an optimum pH value of 7.1 or 9.0, depending on the use of MnATP2- or MgATP2-, respectively, as substrate. Free Mg2+ was an activator at pH values below 9.0. The enzyme was strongly activated by monovalent cations; NH+4 and K+ were the better activators, with apparent Ka values of 0.7 and 1.6 mM, respectively. Partially purified enzymes from cells grown on glucose at 1 or 20 ° had the same properties, including the thermal stability. In both cases 50% of the enzyme activity was lost after preincubation for 10 min at 46 °. The molecular weight was estimated to be about 300000 daltons by gel filtration on Sephadex G-200. The regulatory properties and molecular weight are thus similar to those determined for the pyruvate carboxylases from Pseudomonas citronellolis and Azotobacter vinelandii.