The mode of binding of pyridoxal 5'-phosphate in rabbit muscle glycogen phosphorylase b: circular dichroism and absorption studies.

Large bands absorbing at 251 and 335 nm were observed in the difference circular dichroism and difference absorption spectra of the holo- versus the apo-enzyme of rabbit muscle phosphorylase b. In addition to the previously known band at 335 nm, the band at 251 nm is here assigned to the bound pyridoxal 5'-phosphate (PLP). These observed difference spectra are simulated by the optical properties of the enol-imine form of the Schiff base rather than those of the substituted aldimine. Pyridoxal and salicylaldehyde also bound to apophosphorylase in the same manner as PLP. Since analogs having no hydroxyl group at the ortho position to the aldehyde group did not bind to the apoenzyme, the hydroxyl group is thought to be important in stabilizing the interaction between apophosphorylase and PLP. The PLP bound at Site I of bovine serum albumin, which is known to be one of the PLP-binding proteins absorbing at 330-340 nm, showed two prominent bands at 253 and 335 nm. The circular dichroism and absorption spectra induced by the binding of PLP fit those of the enol-imine of the Schiff base, as in the case of phosphorylase.