Studies of 6-fluoropyridoxal and 6-fluoropyridoxamine 5'-phosphates in cytosolic aspartate aminotransferase

Abstract

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.

15N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution

“…The pK a values for the imino nitrogens of 2a and 3 obtained here correspond closer to the apparent pK a values of external aldimines, which are usually [9][10][11][42][43][44] than to those of internal aldimines, which are in order of 6-7. [45][46][47] The electron-withdrawing effect of the imino function affects the ring nitrogen whose pK a decreases to 5.8, a normal value for pyridine derivatives. [48][49][50] One anticipates an even larger drop in the absence of the doubly negatively charged phosphate group.…”

19F: A Versatile Reporter for Non-Invasive Physiology and Pharmacology Using Magnetic Resonance

“…This affects the electronic environment around fluorine at the 6-position, which is particularly pronounced due to the para location. Extensive literature on modifications of vitamin B 6 suggested that the introduction of electron donating or withdrawing groups at the 4 and 5 positions of the pyridoxol ring could significantly alter the pKa of the 3-phenolic group, and the NMR properties at the 6-position [95,99,100]. From a series of analogs 6-fluoropyridoxamine (6-FPAM) was identified as an enhanced pH indicator (pKa 7.05) [94].…”

Spectroscopic study of the Schiff bases of dodecylamine with pyridoxal 5′-phosphate and 5′-deoxypyridoxal. A model for the Schiff bases of pyridoxal 5′-phosphate in biological systems

“…Information on the state of the coenzyme in the holoenzyme has largely been obtained by using u.v.-visible absorption spectroscopy or spectrofluorimetry. Thus the spectroscopic features of vitamin B6 derivatives (Matsushima & Martell, 1967;Metzler et al, 1973;Harris et al, 1974;Scott et al, 1985) and related Schiff bases (Metzler et al, 1980;Donoso et al, 1986;Morozov et al, 1988) have been widely studied.…”