Substitution of pyridoxal 5 '-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis

D-Serine dehydratase (DSD) is a pyridoxal 5'-phosphate-dependent enzyme tha t catalyzes the conversion of D-serine to pyruvate and ammonia. Spectral st udies of enzyme species where the natural cofactor was substituted by pyrid oxal 5'-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and p yridoxal 5'-phosphate monomethyl ester (PLPMe) were used to gain insight in to the structural basis for binding of cofactor and substrate analogues. PD MP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD a nd PLPMe-DSD are catalytically inactive. The emission spectrum of native DS D when excited at 280 nm shows maxima at 335 and 530 nm, The energy transfe r band at 530 nm is very likely generated as a result of the proximity of T rp-197 to the protonated internal Schiff base, The cofactor analogue-recons tituted DSD species exhibit emission intensities decreasing from PLS-DSD, t o PLPMe-DSD, and PDMP-DSD, when excited at 415 nm. Large increases in fluor escence intensity at 530 (540) nm can be observed for cofactor analogue-rec onstituted DSD in the presence of substrate analogues when excited at 415 n m, In the absence and presence of substrate analogues, virtually identical far UV CD spectra were obtained for all DSD species. The visible CD spectra of native DSD, PDMP-DSD, and PLS-DSD exhibit a band centered on the visibl e absorption maximum with nearly identical intensity. Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species result s in most cases in a decrease or elimination of ellipticity. The results ar e interpreted in terms of local conformational changes and/or changes in th e orientation of the bound cofactor (analogue).