title: A QM/MM study of the L-threonine formation reaction of threonine synthase: implications into the mechanism of the reaction specificity.
abstract: Threonine synthase catalyzes the most complex reaction among the pyridoxal-5'-phosphate (PLP)-dependent enzymes. The important step is the addition of a water molecule to the Cβ-Cα double bond of the PLP-α-aminocrotonate aldimine intermediate. Transaldimination of this intermediate with Lys61 as a side reaction to form α-ketobutyrate competes with the normal addition reaction. We previously found that the phosphate ion released from the O-phospho-l-homoserine substrate plays a critical role in specifically promoting the normal reaction. In order to elucidate the detailed mechanism of this "product-assisted catalysis", we performed comparative QM/MM calculations with an exhaustive search for the lowest-energy-barrier reaction pathways starting from PLP-α-aminocrotonate aldimine intermediate. Satisfactory agreements with the experiment were obtained for the free energy profile and the UV/vis spectra when the PLP pyridine N1 was unprotonated and the phosphate ion was monoprotonated. Contrary to an earlier proposal, the base that abstracts a proton from the attacking water was the ε-amino group of Lys61 rather than the phosphate ion. Nevertheless, the phosphate ion is important for stabilizing the transition state of the normal transaldimination to form l-threonine by making a hydrogen bond with the hydroxy group of the l-threonine moiety. The absence of this interaction may account for the higher energy barrier of the side reaction, and explains the mechanism of the reaction specificity afforded by the phosphate ion product. Additionally, a new mechanism, in which a proton temporarily resides at the phenolate O3' of PLP, was proposed for the transaldimination process, a prerequisite step for the catalysis of all the PLP enzymes.
authors.affiliation: Center for Computational Sciences, University of Tsukuba , Tennodai 1-1-1, Tsukuba 305-8577, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Shoji
authors.forename: Kyohei
authors.initials: K
authors.lastname: Hanaoka
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Ujiie
authors.forename: Wataru
authors.initials: W
authors.lastname: Tanaka
authors.forename: Daiki
authors.initials: D
authors.lastname: Kondo
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Umeda
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Kamoshida
authors.forename: Megumi
authors.initials: M
authors.lastname: Kayanuma
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Kamiya
authors.forename: Kenji
authors.initials: K
authors.lastname: Shiraishi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Machida
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murakawa
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Hayashi
mesh_terms: D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000378:metabolism; D007097:Imines / Q000737:chemistry; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D011789:Quantum Theory; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry*; D014867:Water / Q000737:chemistry
chemical_list: D007097:Imines; D010710:Phosphates; D011522:Protons; D014867:Water; D013912:Threonine; D019757:Carbon-Oxygen Lyases; C023160:threonine synthase