1,3-Proton Transfer of Pyridoxal 5'-Phosphate Schiff Base in the Branched-Chain Aminotransferase: Concerted or Stepwise Mechanism?

Abstract

The branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE) is a pyridoxal 5'-phosphate (PLP) dependent enzyme, and it is essential for the synthesis of the branched-chain amino acids. Ketimine is an important intermediate in the catalytic process. We have investigated the mechanism of ketimine formation and the energy landscape using the multiple computational methods. It is found that the 1,3-proton transfer involved in ketimine formation occurs through a stepwise process rather than a one-step process. Lys204 is identified as a key residue for ligand binding and as a base that abstracts the Cα proton from the PLP-Glu Schiff base, yielding a carbanionic intermediate. The first proton transfer is the rate-limiting step with an energy barrier of 17.8 kcal mol-1. Our study disclosed the detailed pathway of the proton transfer from external aldimine to ketimine, providing novel insights into the catalytic mechanism of other PLP-dependent enzymes.