Redesigning the PheA Domain of Gramicidin Synthetase Leads to a New Understanding of the Enzyme's Mechanism and Selectivity

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• 作者：Brian W. Stevens ; Ryan H. Lilien ; Ivelin Georgiev ; Bruce R. Donald ; Amy C. Anderson
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：December 26, 2006
• 年：2006
• 卷：45
• 期：51
• 页码：15495 - 15504
• 全文大小：259K
• 年卷期：v.45,no.51(December 26, 2006)
• ISSN：1520-4995

文摘

The PheA domain of gramicidin synthetase A, a non-ribosomal peptide synthetase, selectivelybinds phenylalanine along with ATP and Mg²⁺ and catalyzes the formation of an aminoacyl adenylate.In this study, we have used a novel protein redesign algorithm, K^*, to predict mutations in PheA thatshould exhibit improved binding for tyrosine. Interestingly, the introduction of two predicted mutationsto PheA did not significantly improve K_D, as measured by equilibrium fluorescence quenching. However,the mutations improved the specificity of the enzyme for tyrosine (as measured by k_cat/K_M), primarilydriven by a 56-fold improvement in K_M, although the improvement did not make tyrosine the preferredsubstrate over phenylalanine. Using stopped-flow fluorometry, we examined binding of different aminoacid substrates to the wild-type and mutant enzymes in the pre-steady state in order to understand theimprovement in K_M. Through these investigations, it became evident that substrate binding to the wild-type enzyme is more complex than previously described. These experiments show that the wild-typeenzyme binds phenylalanine in a kinetically selective manner; no other amino acids tested appeared tobind the enzyme in the early time frame examined (500 ms). Furthermore, experiments with PheA,phenylalanine, and ATP reveal a two-step binding process, suggesting that the PheA-ATP-phenylalaninecomplex may undergo a conformational change toward a catalytically relevant intermediate on the pathwayto adenylation; experiments with PheA, phenylalanine, and other nucleotides exhibit only a one-step bindingprocess. The improvement in K_M for the mutant enzyme toward tyrosine, as predicted by K^*, may indicatethat redesigning the side-chain binding pocket allows the substrate backbone to adopt productiveconformations for catalysis but that further improvements may be afforded by modeling an enzyme:ATP:substrate complex, which is capable of undergoing conformational change.