Studies on tryptophan residues of fructose-6-phosphate-2-kinase

Abstract

The results of ultraviolet spectroscopy of NBS titrating PFK-2 indicate that the enzyme has four tryptophase residues. Among them two are essential. There is a strong emission fluorescence at the 335 nm region when chicken liver PFK-2 excited with UV light as 295 nm. This fluorescence can specifically reflect the states of tryptophane residues of the enzyme. Substrate Fru6P does not affect the fluorescence but ATP causes a quenching of the fluorescence. The maximum quenching is about 30%. The binding of ATP to PFK-2 has been monitored by fluorescence. It has been obtained that K-i is 0.033 mmol/L in the low ATP range, while it is 0.23 mmol/L in the high ATP concentration range. This illustrates that the binding of ATP to the enzyme possesses a negative cooperativity. Both Mg-2+ and inorganic phosphate are activators which reduce the K-i values. Fru6P and Fru2, 6P-2 have no effects on K-i. The binding at ATP induces a relatively large change in the conformation of the enzyme. It has been noted that in the presence of ATP the inactivation rate of the enzyme by NBS slightly increases. From the quenching of fluorescence, the Stern-Volmer constant (K-sv) is 4.0 either in the presence or in the absence of ATP as measured by using acrylamide to quench the tryptophane fluoresence of the enzyme.