Purification, Properties, and a Possible Mechanism for Pyridoxal Kinase from Bovine Brain

Pyridoxal kinase catalyzes the formation of pyridoxal phosphate, a coenzyme required for many metabolic reactions. A 2000-fold purification of the enzyme from bovine brain has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration, and affinity chromatography. Polyacrylamide gel electrophoresis at pH 9.5 and 7.5 indicates that the final product is 70 to 75% homogeneous. From sedimentation equilibrium analysis, the highly purified preparation appears to be monodisperse, and a molecular weight estimate of 80,000 has been made. A sharp pH optimum at pH 6.0 has been observed. Of the divalent metal ions studied, Zn+2 is the most effective in catalyzing the formation of pyridoxal phosphate from ATP and pyridoxal; the order of activation is Zn+2 g Co+2 g Mn+2 g Mg+2 g Fe+2. The stability constants for ZnHATP- and ZnATP-2 have been obtained at 0.3 ionic strength and 25° in the absence of Na+ or K+ by the pH titration method. The values are 1050 m-1 for ZnHATP- and 195,000 m-1 for ZnATP-2. An initial velocity study using pyridoxal as the variable substrate and ZnATP-2 as the fixed substrate gave a family of lines intersecting to the left of the vertical axis. Such an intersecting pattern is compatible with a sequential mechanism in which both pyridoxal and ZnATP-2 must add to the enzyme before pyridoxal phosphate or ADP is released.