Mechanism of action of D-serine dehydratase. Identification of a transient intermediate

Abstract

Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2.degree. C show that during the steady-state course of D-serine conversion the absorption maximum of the Schiff base of the cofactor pyridoxal 5''-phosphate (pyridoxal-P) is shifted from 415 to 442 nm. The progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320-500 nm. A point by point construction of successive spectra from these stopped-flow traces at various time intervals after the start of reaction resulted in a series of consecutive spectra exhibiting 2 isobestic points at 353 and 419 nm. The half-time of the absorbance changes occurring at 330 and 455 nm was 6.5 ms, suggesting the observation of a single, enzyme-bound intermediate. The spectral data with substrate and inhibitors provide evidence that the intermediate is the Schiff base of .alpha.-aminoacrylate and pyridoxal-P. The proposed assignment is strongly supported by experiments of apodehydratase with transient-state analogs which exhibit a similar absorbance shift on binding to apoenzyme. The phosphate group of the substrate-pyridoxal-P complex probably serves as the main anchoring point during catalysis. A reaction mechanism of the D-serine dehydratase is presented.