Structural changes accompanying the removal of pyridoxal 5'-phosphate from phosphorylase b.

The changes in physical properties accompanying the removal of pyridoxal 5'-phosphate from glycogen phosphorylase b have been examined. The apoenzyme retains a high degree of structural rigidity, as determined from the time decay of anisotropy. The bulk of the secondary structure remains intact, although a significant change in circular dichroism indicates some degree of alteration. The mobility of a sulfhydryl-linked spin label increases. The restoration of pyridoxal 5'-phosphate reverses this effect, with indication of interaction between subunits. One or more new binding sites for 1-anilinonaphthalene-8-sulfonate appear for the apoenzyme. The kinetics of the recombination of pyridoxal 5'-phosphate with the apoenzyme, as monitored by difference spectra, indicate a high activation energy for the process. The apoenzyme is a reversibly associating system at 20-30 degrees C, pH 7.0.