Catalysis by a De Novo Zinc-Mediated Protein Interface: Implications for Natural Enzyme Evolution and Rational Enzyme Engineering

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• 作者：Bryan S. Der ; David R. Edwards ; Brian Kuhlman
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：May 8, 2012
• 年：2012
• 卷：51
• 期：18
• 页码：3933-3940
• 全文大小：367K
• 年卷期：v.51,no.18(May 8, 2012)
• ISSN：1520-4995

文摘

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein鈥損rotein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10⁵ and a k_cat/K_M of 630 M^鈥? s^鈥? and 4-nitrophenyl phosphate with a rate acceleration of 10⁴ and a k_cat/K_M of 14 M^鈥? s^鈥?. These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein鈥損rotein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.