A QM/MM Study of the l-Threonine Formation Reaction of Threonine Synthase: Implications into the Mechanism of the Reaction Specificity

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• 作者：Mitsuo Shoji ; Kyohei Hanaoka ; Yuzuru Ujiie ; Wataru Tanaka ; Daiki Kondo ; Hiroaki Umeda ; Yoshikazu Kamoshida ; Megumi Kayanuma ; Katsumasa Kamiya ; Kenji Shiraishi ; Yasuhiro Machida ; Takeshi Murakawa ; Hideyuki Hayashi
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：March 26, 2014
• 年：2014
• 卷：136
• 期：12
• 页码：4525-4533
• 全文大小：460K
• 年卷期：v.136,no.12(March 26, 2014)
• ISSN：1520-5126

文摘

Threonine synthase catalyzes the most complex reaction among the pyridoxal-5鈥?phosphate (PLP)-dependent enzymes. The important step is the addition of a water molecule to the C尾鈥揅伪 double bond of the PLP鈭捨?aminocrotonate aldimine intermediate. Transaldimination of this intermediate with Lys61 as a side reaction to form 伪-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 鈥減roduct-assisted catalysis鈥? we performed comparative QM/MM calculations with an exhaustive search for the lowest-energy-barrier reaction pathways starting from PLP鈭捨?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 蔚-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鈥?of PLP, was proposed for the transaldimination process, a prerequisite step for the catalysis of all the PLP enzymes.