J. Biol. Chem., Vol. 260, Issue 5, 2709-2714, Mar, 1985
Use of 6-fluoroderivatives of pyridoxal and pyridoxal phosphate in the study of the coenzyme function in glycogen phosphorylase
YC Chang and DJ Graves
6-Fluoropyridoxal phosphate (6-FPLP) has been synthesized. Its properties
were studied, and it was used, along with 6-fluoropyridoxal (6-FPAL), to
reconstitute apophosphorylase b. Kinetic studies of the resulting enzymes
showed that phosphorylases reconstituted with 6-FPLP and 6-FPAL have
characteristics similar to those of native and pyridoxal enzymes,
respectively, except that the former two enzymes have lower Vmax values.
19F NMR and UV spectra of 6-FPLP phosphorylase showed that the coenzyme
forms a neutral enolimine Schiff base. Because the UV and fluorescence
spectra of 6-FPLP phosphorylase are comparable to those obtained with
native phosphorylase, it further confirms the postulate that pyridoxal
phosphate forms a neutral enolimine Schiff base in phosphorylase. The
results suggest that the 3-OH group is protonated and the pyridine nitrogen
unprotonated in both 6-FPLP phosphorylase and native enzyme. 19F NMR study
of 6-FPLP- and 6-FPAL- reconstituted phosphorylases in the inactive and
active states indicates that the protein structure near the coenzyme
binding site undergoes certain changes when these enzymes are activated by
the substrates and AMP. The comparison of the properties of 6-FPLP-
reconstituted and native phosphorylases implies that the ring nitrogen of
the coenzyme PLP in phosphorylase may interact with the protein during
catalysis, and this interaction is important for efficient catalysis by
phosphorylase.
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