Tyrosine Phenol Lyase

Abstract

Abstract Crystalline l-tyrosine phenol lyase of Escherichia intermedia A-21 is inactive in the absence of added pyridoxal phosphate. Half-maximal enzymatic activity is obtained at a concentration of 1.3 x 10-6 m pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 430 mµ. Holotyrosine phenol lyase requires K+ or NH4+ for its maximal activity, but Na+ is inactive. When K+ is replaced by Na+, a small decrease in absorbance at 430 mµ is observed. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis and spectrophotometric titration to be 2 moles per mole of enzyme. Reduction of holoenzyme with sodium borohydride results in a shift of the absorption peak at 430 mµ to 336 mµ. e-Pyridoxyllysine was isolated from the acid hydrolysate of the reduced holoenzyme by paper chromatography and electrophoresis. Addition of the substrate, l-tyrosine, or the competitive inhibitor, l-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mµ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.