Synthesis of a cationic pyridoxamine conjugation reagent and application to the mechanistic analysis of an artificial transaminase

An N-methylated, cationic pyridoxamine conjugation reagent was synthesized and tethered via a disulfide bond to a cysteine residue inside the cavity o f intestinal fatty acid binding protein. The conjugate was characterized an d the kinetic parameters compared to its nonmethylated pyridoxamine analogu e. Kinetic isotope effects were used for further mechanistic analysis. Take n together, these experiments suggest that a step distinct from deprotonati on of the ketimine in the pyridoxamine to pyridoxal reaction is what limits the rate of the artificial transaminase IFABP-Px. However, the internal en ergetics of reactions catalyzed by the conjugate containing the N-methylate d cofactor appear to be different suggesting that the MPx reagent will be u seful in future experiments designed to alter the catalytic properties of s emisynthetic transaminases. (C) 2000 Elsevier Science Ltd. All rights reser ved.