Mechanism of action of D-serine dehydratase. Identification of a transient intermediate.
Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2 degrees 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. Furthermore, the progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320 to 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 two isobestic points at 353 and 419 nm. The half-time of the absorbance changes occurring at 330 and 455 nm was found to be 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 analogues which exhibit a similar absorbance shift on binding to apoenzyme. Moreover, these results suggest that the phosphate group of the substrate--pyridoxal-P complex serves as the main anchoring point during catalysis. A reaction mechanism of the D-serine dehydratase is presented.[1]References
- Mechanism of action of D-serine dehydratase. Identification of a transient intermediate. Schnackerz, K.D., Ehrlich, J.H., Giesemann, W., Reed, T.A. Biochemistry (1979) [Pubmed]
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