Isolation and properties of crystalline quinolinate phosphoribosyltransferase from hog kidney

Crystalline quinolinate phosphoribosyltransferase (nicotinatenucleotide: pyrophosphate phosphoribosyltransferase (carboxylating), EC 2.4.2.19) was isolated from hog kidney and compared with the same enzyme prepared from hog liver. The enzyme preparation was homogeneous as shown by polyacrylamide gel electrophoresis and ultracentrifugation analysis. The enzyme had a molecular weight of 220 000 and the subunit 35 000. The physicochemical properties of the enzyme were: sedimentation coefficient (S200,W), 7.75 . 10(-13) s; difussion coefficient (D200,W), 5.04 . 10(-7) cm2/s; Stokes radius, 62.05 A, frictional ratio (f/f0), 1.62 and isoelectric point (pI), 4.5. The enzyme was stable at 37 degrees C for 30 min between pH 4.5 and 9.5. Enzyme activity was inhibited by various carboxylic acids; however, this inhibition was reversed by raising the Mg2+ concentration. Optimum pH was 5.5, and no detectable amounts of Mg2+, Mn2+, Fe2+, Cu2+, Zn2+ and Ca2+ were found by atomic absorption spectrophotometry. The enzyme was found to contain sugar. Mg2+ was completely replaceable by Mn2+. The reaction mechanism of this enzyme was suggested to be of the 'ping-pong' type. Km values of quinolinic acid and 5-phosphoribosyl 1-pyrophosphate were 4 . 10(-5) and 1.4 . 10(-4) M, respectively.