Physical-chemical studies on the pyridoxal phosphate binding site in sodium borohydride-reduced and native phosphorylase.

NaBH4-reduced phosphorylase b is a catalytically active species which retains many of the properties of the native enzyme. One property that is strikingly different, however, is the spectral behavior of bound pyridoxal phosphate. At pH 7.0, the pyridoxal phosphate shows essentially no absorption at 333 nm, where pyridoxal phsophate in the native enzyme absorbs; absorption at 333 mn returns upon lowering the pH. The 333 run band at low pH in the reduced enzyme was found to be optically active, (AA/A) 333 nm s 0.7 X 10-a. Circular dichroism of the reduced phosphorylase b at pH 7.0 indicated that no new band was formed above 300 nm; the ultraviolet spectrum also provided no evidence for a band above 300 mn. Guanidine~HCl (4.25 M) and sodium dodecyl sulfate (0.3%) caused the appearance of a band near 330 nm at pH 6.8. Difference spectra of reduced phosphorylase b (&sodium dodecyl sulfate; pH 5 versus pH 7) indicated that the hidden band must be below 300 nm. AMP (0.01 M) was shown to cause partial reversal of the hidden band to 333 nm conversion at pH 6.0; ATP was also effective. Substrates and AMP had no effect on the spectrum of the reduced phosphorylase b at pH 6.8. Ultracentrifugal studies showed that native and NaBHB-reduced enzyme have difYerent conformations. The data were interpreted to mean that the 333 mn and hidden band forms of NaBH*-reduced phosphorylase represent different enzyme conformations. The hidden band form is thought to represent a form of pyridoxal phosphate in which the pyridinium nitrogen is unprotonated and the 3-hydroxyl group undissociated or strongly hydrogen bonded; the 333 mn species is believed to be a dipolar form of pyridoxal phosphate which is exposed to solvent. Spectral properties of enzyme-bound pyridoxal phosphate