title: Chemical Mechanism of the Branched-Chain Aminotransferase IlvE from Mycobacterium tuberculosis.
abstract: The biosynthetic pathway of the branched-chain amino acids is essential for Mycobacterium tuberculosis growth and survival. We report here the kinetic and chemical mechanism of the pyridoxal 5'-phosphate (PLP)-dependent branched-chain aminotransferase, IlvE, from M. tuberculosis (MtIlvE). This enzyme is responsible for the final step of the synthesis of the branched-chain amino acids isoleucine, leucine, and valine. As seen in other aminotransferases, MtIlvE displays a ping-pong kinetic mechanism. pK values were identified from the pH dependence on V as well as V/K, indicating that the phosphate ester of the PLP cofactor, and the α-amino group from l-glutamate and the active site Lys204, play roles in acid-base catalysis and binding, respectively. An intrinsic primary kinetic isotope effect was identified for the α-C-H bond cleavage of l-glutamate. Large solvent kinetic isotope effect values for the ping and pong half-reactions were also identified. The absence of a quininoid intermediate in combination with theDkobsin our multiple kinetic isotope effects under single-turnover conditions suggests a concerted type of mechanism. The deprotonation of C2 of l-glutamate and the protonation of C4' of the PLP cofactor happen synchronously in the ping half-reaction. A chemical mechanism is proposed on the basis of the results obtained here.
authors: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Tathyana M
authors: TM
authors: Amorim Franco
authors: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Subray
authors: S
authors: Hegde
authors: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: John S
authors: JS
authors: Blanchard
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D020134:Catalytic Domain; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism; D014633:Valine / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007532:Isoleucine; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase; D007930:Leucine; D014633:Valine; D008239:Lysine