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 that catalyzes the conversion of D-serine to pyruvate and ammonia. Spectral studies of enzyme species where the natural cofactor was substituted by pyridoxal 5'-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and pyridoxal 5'-phosphate monomethyl ester (PLPMe) were used to gain insight into the structural basis for binding of cofactor and substrate analogues. PDMP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD and PLPMe-DSD are catalytically inactive. The emission spectrum of native DSD when excited at 280 nm shows maxima at 335 and 530 nm. The energy transfer band at 530 nm is very likely generated as a result of the proximity of Trp-197 to the protonated internal Schiff base. The cofactor analogue-reconstituted DSD species exhibit emission intensities decreasing from PLS-DSD, to PLPMe-DSD, and PDMP-DSD, when excited at 415 nm. Large increases in fluorescence intensity at 530 (540) nm can be observed for cofactor analogue-reconstituted DSD in the presence of substrate analogues when excited at 415 nm. 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 visible absorption maximum with nearly identical intensity. Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species results in most cases in a decrease or elimination of ellipticity. The results are interpreted in terms of local conformational changes and/or changes in the orientation of the bound cofactor (analogue).
Schnackerz
K D
KD
Theodor-Boveri Institut für Biowissenschaften, Physiologische Chemie I, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany. schnacke@biozentrum.uni-wuerzburg.de
Tai
C H
CH
Pötsch
R K
RK
Cook
P F
PF
D000596:Amino Acids / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D012695:L-Serine Dehydratase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry*
D000596:Amino Acids; D011732:Pyridoxal Phosphate; D012695:L-Serine Dehydratase