PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE ADP-GLUCOSE PYROPHOSPHORYLASE FROM THERMUS-CALDOPHILUS GK-24

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has b een purified from Thermus caldophilus GK-24 to homogeneity by chromato graphic methods, including gel filtration and ion-exchange and affinit y chromatography. The specific activity of the enzyme was enriched 134 .8-fold with a recovery of 10.5%. The purified enzyme was a single ban d by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric stru cture of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indi cating that the structure of the enzyme is different from the heterote trameric structures of higher-plant AGPases. The enzyme was most activ e at pH 6.0. The activity was maximal at 73-78 degrees C and its half- life was 30 min at 95 degrees C. Kinetic and regulatory properties wer e characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructo se 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effect ive activators, of which fructose 1,6-bisphosphate was the most effect ive. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucos e l-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino aci d composition was the existence of the hydrophobic and Ala + Gly resid ues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently s imilar to those of AGPases from other bacterial and plant sources, sug gesting that the enzymes are structurally related.