Effects of steroid and nonsteroid metabolites on enzyme conformation and pyridoxal phosphate binding.

An understanding of the factors that influence the binding of pyridoxal phosphate (PLP) to the various tissue constituents is of major importance for an understanding of the metabolism, distribution and function of that coenzyme in mammalian systems. The main objectives of this paper are to review some studies pertaining to PLP binding and to present some of our more recent results that seem to bear on their biological relevance. Our studies of PLP binding began with the discovery in 1957 that the kynurenine transaminase in freshly-prepared rat kidney homogenates was inactivated rapidly during incubation at 37 degrees C. and that added PLP restored the activity.' We concluded that the inactivation reflected the loss of PLP from the enzyme and that factors which influenced the rate of loss did so by influencing the binding of PLP. Thus it was found that the dissociation was accelerated by lowering the pH and by increasing the concentration of inorganic phosphate (Pi) in the incubation medium. a-Ketoglutarate, one of the substrates, effectively prevented the dissociation when present at substrate levels. Similar actions of Pi were subsequently observed with other pyridoxal enzymes." Scardi and associates2 reported that Pi promoted the dissociation of the coenzyme from glutarnate-aspartate transaminase but only after it was converted to pyridoxamine phosphate by reaction with an amino acid substrate. The relatively greater binding stability of the pyridoxal form is usually attributed to the binding of the aldehyde group via a Schiff base linkage to an €-amino group of the apoen~yme.~ Transamination with the amino acid substrate breaks this linkage by converting the PLP to pyridoxamine phosphate.6 Presumably the Pi then displaces the ester phosphate group of the coenzyme from a cationic binding site of the apoenzyme, allowing ready dissociation. The stabilization of PLP binding by a-ketoglutarate, first observed in our studies of kynurenine transaminase, is thus probably characteristic of the transaminases. In addition, several pyridoxal enzymes which exhibit transarninase activity only as a minor function are activated and inactivated in a similar way. Novogrodsky and Meister' found that bacterial aspartate P-decarboxylase was inactivated by incubation with various L-amino acids and that the inactivation was prevented by analogous a-keto acids and was reversed by PLP. Enzymic and spectrophotometric studies indicated that the inactivation was due to the formation of pyridoxamine phosphate by transamination, followed by dissociation of that coenzyme from the apodecarboxylase. A similar action of amino acid and keto acid substrates was observed by Schirch and Jenkinss with serine transhydroxymethylase. That enzyme underwent transamination of the enzyme-bound PLP with palanine to yield pyridoxamine phosphate, the apoenzyme, and pyruvate. These studies emphasize the much greater binding stability of PLP as compared with pyridoxamine phosphate. However, the PLP can be removed from