Some properties of the pyruvate carboxylase from Pseudomonas fluorescens.
The pyruvate carboxylase of Pseudonomas fluorescens was purified 160-fold  from cells grown on glucose at 20 degrees C. 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 HCO3 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; NH4+ 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 degrees C had the same properties, including the thermal stability. In both cases 50% of the enzyme activity was lost after pre-incubation for 10 min at 46 degrees C. 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.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000494:pharmacology; D001360:Avidin / Q000494:pharmacology; D001710:Biotin / Q000494:pharmacology; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D013696:Temperature
D011773:Pyruvates; D001360:Avidin; D001224:Aspartic Acid; D008345:Manganese; D000244:Adenosine Diphosphate; D001710:Biotin; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D011766:Pyruvate Carboxylase; D008274:Magnesium; D005947:Glucose