d-Serine Dehydratase from Escherichia coli

Abstract d-Serine dehydratase from Escherichia coli has two distinct binding sites for pyridoxal 5'-phosphate; a high affinity site (Kdiss 0.03 µm) which binds the catalytically essential coenzyme and a low affinity site (Kdiss near 1000 µm) which binds pyridoxal-P, pyridoxal, or 5-deoxypyridoxal approximately equally, with resultant inhibition of enzymatic activity. Following borohydride reduction, carboxymethylation and chymotryptic digestion of the holoenzyme, a single non-apeptide containing the N6-pyridoxyllysine residue, and thus corresponding to a portion of the high affinity coenzyme binding site was isolated by two different chromatographic procedures and shown to have the sequence: Ser-(Pxy)Lys-Gly-Arg-Ile-Asn-Lys-Ala-Thr. The peptide differs from the corresponding coenzyme-binding site peptides isolated from other pyridoxal-P-dependent enzymes in its highly basic and relatively hydrophilic character. When borohydride reduction of d-serine holodehydratase was carried out in the presence of an inhibitory concentration (7.5 mm) of pyridoxal-P, one additional major pyridoxylpeptide, corresponding to a portion of the low affinity pyridoxal-binding site was isolated. The sequence of this inhibitory site peptide was determined to be: Val-(Pxy)Lys-Ala-Gly-Ala-Phe.