PYRIDOXAL 5'-PHOSPHATE IN ALPHA-GLUCAN PHOSPHORYLASE FROM POTATO

1. 1. Some chemical properties of α-glucan phosphorylase (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) from potato were studied, in particular, pyridoxal 5′-phosphate bound to the enzyme protein. 2. 2. The bound pyridoxal 5′-phosphate in α-glucan phosphorylase from potato changes to a form absorbing at 390 mμ at an alkaline pH. Pyridoxal 5′-phosphate was removed from the enzyme protein by incubating in 4.2 M urea, with a concomitant loss of enzyme activity. The presence of 0.02 M NaBH4 protected the phosphorylase from inctivation by urea, but the bound pyridoxal 5′-phosphate was not attached to the enzyme under these conditions. 3. 3. The binding of pyridoxal 5′-phosphate to the enzyme protein was accomplished by reduction with 0.06 M NaBH4 at pH 6.0, or with 0.02 M NaBH4 at alkaline pH. 4. 4. The reduced enzyme protein lost its absorption peak at 330 mμ, and, in contrast to the native enzyme, showed a positive blue color in the dichloroquinone chloroimide test and a positive orange color in the p-amino acetophenone test. It was concluded that the 3-hydroxyl group of pyrdixoal 5′-phosphate was linked to the native enzyme in some way possibly by a hydrogen bond. 5. 5. In the course of reduction, decrease of absorption at 330 mμ was not found to parallel loss of enzyme activity. 6. 6. On reduction with 0.06 M NaBH4, the addition of l-lysine or Tris protected the bound pyridoxal 5′-phosphate against reductive fixation, and caused a loss of enzymic activity by some unknown mechanism. © 1969.