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 of intestinal fatty acid binding protein. The conjugate was characterized and the kinetic parameters compared to its nonmethylated pyridoxamine analogue. Kinetic isotope effects were used for further mechanistic analysis. Taken together, these experiments suggest that a step distinct from deprotonation of the ketimine in the pyridoxamine to pyridoxal reaction is what limits the rate of the artificial transaminase IFABP-Px. However, the internal energetics of reactions catalyzed by the conjugate containing the N-methylated cofactor appear to be different suggesting that the MPx reagent will be useful in future experiments designed to alter the catalytic properties of semisynthetic transaminases.
Kuang
H
H
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Häring
D
D
Qi
D
D
Mazhary
A
A
Distefano
M D
MD
D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002412:Cations; D050556:Fatty Acid-Binding Proteins; D007700:Kinetics; D008958:Models, Molecular; D018716:Molecular Mimicry; D009363:Neoplasm Proteins; D011733:Pyridoxamine / Q000737:chemistry* / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; D002412:Cations; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D011733:Pyridoxamine; D000637:Transaminases