A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transminase enzymes

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C α and C-4'positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular p K a of the monoprotic base. Thus the ε-ammonium group of the internal aldimine-forming lysine residue operates at C-4'- si -face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C α from the 4'- si -face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldim ination processes and as a binding group for the α-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. — S— X— H — K — ) with that for aspartate aminotransferase (e.g. — S— X — A— K— ) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data. A similar model for serine hydroxymethyltransferase suggested that previous reports of stereochemical infidelity with decarboxylation substrates were incorrect. A series of careful experiments confirmed this. Hence, no actual examples of non-stereospecific α-amino acid decarboxylation by pyridoxal enzymes exist.