Purification and Properties of d-Serine Dehydrase from Escherichia coli

Abstract

Abstract A procedure is described for obtaining crystalline d-serine dehydrase from a mutant of Escherichia coli which produces this enzyme constitutively. The enzyme appears pure by ultracentrifugal and electrophoretic criteria. In sucrose gradients, serine dehydrase sediments to the same position as horseradish peroxidase, indicating a molecular weight of about 40,000. This corresponds closely to its combining weight of 42,800 per pyridoxal phosphate. Its absorption maximum at 410 mµ due to bound pyridoxal phosphate is not materially changed by interaction with substrate or by variation in pH between 6.0 and 9.0. d-Serine and d-threonine are the only substrates found; O-methylserine is a potent competitive inhibitor. In the presence of suitable protective agents, the enzyme can be resolved completely by dialysis against d- or l-cysteine, and reactivated by addition of pyridoxal phosphate. Tris buffer inactivates the enzyme; this inactivation is prevented by the presence of sufficient K+ or NH4+, and less effectively by Na+ or pyridoxal phosphate.