Conservation of Mechanism in Three Chorismate-Utilizing Enzymes

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• 作者：Ze He ; Kimberly D. Stigers Lavoie ; Paul A. Bartlett ; and Michael D. Toney
• 刊名：Journal of the American Chemical Society
• 出版年：2004
• 出版时间：March 3, 2004
• 年：2004
• 卷：126
• 期：8
• 页码：2378 - 2385
• 全文大小：195K
• 年卷期：v.126,no.8(March 3, 2004)
• ISSN：1520-5126

文摘

Chorismate is the end-product of the shikimate pathway for biosynthesis of carbocyclic aromaticcompounds in plants, bacteria, fungi, and some parasites. Anthranilate synthase (AS), 4-amino-4-deoxychorismate synthase (ADCS), and isochorismate synthase (IS) are homologous enzymes that carryout the initial transformations on chorismate in the biosynthesis of tryptophan, p-aminobenzoate, andenterobactin, respectively, and are expected to share a common mechanism. Poor binding to ADCS oftwo potential transition state analogues for addition of a nucleophile to C6 of chorismate implies that it, likeAS and IS, initiates reaction by addition of a nucleophile to C2. Molecular modeling based on the X-raystructures of AS and ADCS suggests that the active site residue K274 is the nucleophile employed byADCS to initiate the reaction, forming a covalent intermediate. The K274A and K274R mutants were shownto have 265- and 640-fold reduced k_cat values when PabA (the cognate amidotransferase) + glutamineare used as the nitrogen source. Under conditions of saturating chorismate and NH₄⁺, ADCS and theK274A mutant have identical k_cat values, suggesting the participation of NH₄⁺ as a rescue agent. Suchparticipation was confirmed by the buildup of 2-amino-2-deoxyisochorismate in the reactions of the K274Amutant but not ADCS, when either NH₄⁺ or PabA + glutamine is used as the nitrogen source. Additionally,the inclusion of ethylamine in the reactions of K274A yields the N-ethyl derivative of 2-amino-2-deoxyisochorismate. A unifying mechanism for AS, ADCS, and IS entailing nucleophile addition to C2 ofchorismate in an S_N2' ' process is proposed.