MODULATION OF THE CATALYTIC ACTIVITY OF BRAIN SUCCINIC SEMIALDEHYDE REDUCTASE BY REACTION WITH PYRIDOXAL 5'-PHOSPHATE

An NADPK-dependent succinic semialdehyde reductase from bovine brain w as inactivated by pyridoxal 5'-phosphate. Spectral evidence is present ed to indicate that the inactivation proceeds through formation of a S chiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosp hopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phos phate. After tryptic digestion of the enzyme modified with pyridoxal 5 '-phosphate in the presence and absence of NADPH followed by [H-3]NaBH 4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region co ntaining the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing resi due, however, can be designated as a phosphopyridoxyl lysine as interp reted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reducta se is modulated by binding of pyridoxal 5'-phosphate to a specific lys yl residue at or near the coenzyme-binding site of the protein.