RECONSTITUTION OF MUSCLE GLYCOGEN-PHOSPHORYLASE-B FROM APOENZYME AND PYRIDOXAL-5'-PHOSPHATE OR ITS ANALOGS, INTERACTION OF APOPHOSPHORYLASEAND THE RECONSTITUTED ENZYME WITH SPECIFIC LIGANDS

Sedimentation methods were used to study the effects of modification o f pyridoxal-5'-phosphate (PLP) at position 5 on the affinity of recons tituted glycogen phosphorylase b from rabbit skeletal muscles for the substrate (glycogen) and the allosteric inhibitor (FMN) as well as on the self-association of the enzyme induced by AMP. Reconstituted phosp horylase b was obtained with PLP analogs containing at position 5 resi dues -CH2-CH2-COOH (analog I), trans-CH=CH-COOH (analog II), or -C=C-C OOH (analog III). Reconstitution of phosphorylase b was shown to be ac companied by the recovery of the quaternary structure of the enzyme. P hosphorylase b reconstituted with PLP or analogs I, II, or III is prac tically indistinguishable from the native enzyme in its affinity for g lycogen. Replacing the native coenzyme in the phosphorylase molecule w ith any one of the tested PLP analogs leads to lower enzyme affinity f or FMN. The microscopic dissociation constants for the FMN-enzyme comp lexes rise in the series enzyme . I < enzyme . II < enzyme . III. Phos phorylase b reconstituted with analogs I, II, or III differs substanti ally from the native enzyme in its ability for self-association in the presence of 1 mM AMP; the reconstituted enzyme exists exclusively in the tetrameric form.