Substitution of pyridoxal 5 '-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis
Kd. Schnackerz et al., Substitution of pyridoxal 5 '-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis, J BIOL CHEM, 274(52), 1999, pp. 36935-36943
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).