D-glucosaminate dehydratase: spectrometric properties of the enzyme-bound pyridoxal 5'-phosphate.
The holoenzyme of D-glucosaminate dehydratase [EC 4.2.1.26] from Agrobacterium radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm. The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme. The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm). The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm. Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity. The occurrence of epsilon-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose epsilon-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base. The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively. The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two Km values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme.
D002942:Circular Dichroism; D006836:Hydro-Lyases / Q000302:isolation & purification*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000302:isolation & purification*; D012231:Rhizobium / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D011732:Pyridoxal Phosphate; D006836:Hydro-Lyases; C018870:aminodeoxygluconate dehydratase