Essential Role of Monovalent Cations in the Firm Binding of Pyridoxal 5′‐Phosphate to Tryptophanase and β‐Tyrosinase

Abstract

Roles of inorganic monovalent cations in tryptophanase of Escherichia coli B and β‐tyrosinase of Escherichia intermedia were studied. Like tryptophanase, β‐tyrosinase absolutely required certain monovalent cations for catalytic activity. The cation selectively of β‐tyrosinase was quite similar to that of tryptophanase; K+, NH4+ and Rb+ were the most effective as cofactor, and Li+ and Na+ were essentially inactive. Cs+ was partially active in the β‐tyrosinase reaction but essentially inactive in the tryptophanase reaction. A close relationship was observed between their cofactor activities and crystal ionic radii. It was established in β‐tyrosinase as well as in tryptophanase that K+ is absolutely required for conversion of the apoenzyme‐pyridoxal 5′‐phosphate complex into the functional form. Effects of monovalent cations and substrates on formation and resolution of the holoenzymes of tryptophanase and β‐tyrosinase were investigated by a Sephadex G‐25 gel filtration method. In both cases, highly active monovalent cations facilitated the formation of the tight enzyme‐pyridoxal 5′‐phosphate complex (holoenzyme) and retarded its resolution, and the reverse was the case for the inactive cations. Upon Sephadex G‐25 gel filtration using triethanolamine‐HCI buffer, the dissociation of pyridoxal 5′‐phosphate from holotryptophanase proceeded according to the first‐order kinetics. Affinities of Na+‐tryptophanase for both coenzyme and substrate were found to be much lower than those of the K+‐enzyme. Thus, it can be concluded that certain monovalent cations play an essential role in formation and maintenance of the firm binding of pyridoxal 5′‐phosphate to the apoenzyme leading to the catalytically active holoenzyme.