Separation and evaluation of the covalent and noncovalent interactions which contribute to the binding of pyridoxal 5'-phosphate to D-serine apodehydratase.
The equilibrium constant (KX) for the reaction D-serine dehydratase + pyridoxamine-P in equilibrium KX D-serine apodehydratase: pyridoxamine-P + pyridoxal-P was determined.  At 25 degreees, pH 7.80, KX increases from 5.4 times 10-minus 5 to 21 times 10-minus 5 as T/2 is increased from 0.33 to 0.66.  A value of 1.3 times 10-minus 4 M at 25 degrees, pH 7.80, T/2 0.33 for the equilibrium constant (KPMP) for dissociation of pyridoxamine-P from D-serine apodehydratase was determined from the ratio of the equilibrium constant for dissociation of pyridoxal-P from holoenzyme to KX.  Pyridoxamine-P and the thiazolidine, formed from pyridoxal-P and cysteine, were found to have similar affinities for D-serine apodehydratase.  Using the affinities of these derivatives as a measure of the noncovalent interactions between cofactor and protein, it was possible to estimate the contribution of the Schiff base linkage to the stability of the complex formed between pyridoxal-P and protein.  The covalent Schiff base linkage in the holoenzyme was found to be no more stable than the Schiff base linkage formed between 6-aminocaproic acid and pyridoxal-P.  The contribution of noncovalent interactions to the stability of the cofactor-protein complex was shown to be at least 20 to 40 times greater than the contribution of the covalent Schiff base linkage.
D002850:Chromatography, Gel; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011733:Pyridoxamine / Q000378:metabolism*; D012545:Schiff Bases; D013056:Spectrophotometry, Ultraviolet
D009943:Organophosphorus Compounds; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D011189:Potassium Chloride; D012695:L-Serine Dehydratase