Enzyme-substrate interactions modulating protonation and tautomerization states of the aldimines of pyridoxal enzymes

Abstract

The mechanism of activation of PLP-Lys aldimines of Aspartate aminotransferase (AspAT), aromatic amino acid aminotransferase (ArAT), and aromatic L-amino acid decarboxylase (AADC) to the ketoenamine form was studied. In AspAT and ArAT the aldimines exist as the nonprotonated form. Upon binding of substrate amino acids, the pK a of these aldimines are increased and the aldimines become the protonated, ketoenamine form, which is considered to be favorable for transaldimination. The increase in pK a by binding of amino acids was proved by mutagenesis studies to be mediated mainly by interaction of α-carboxylate group of the substrate and Arg386 of the enzymes. In AADC, the aldimine is protonated, but it exists as the enolimine tautomer and is not favorable for transaldimination. In the presence of a substrate amino acid, it undergoes tautomerization to the ketoenamine form. Pyridoxal enzymes show a variety of spectra, and PLP-Lys aldimines exist as several protonated/deprotonated forms. However, it is proposed that all these forms are converted to the protonated, ketoenamine form upon binding of substrate amino acids, either by altering the pK a values of the PLP-Lys aldimines or by changing the polarity of the microenvironment around the aldimines.