Light Samuel H, Cahoon Laty A, Mahasenan Kiran V, Lee Mijoon, Boggess Bill, Halavaty Andrei S, Mobashery Shahriar, Freitag Nancy E, Anderson Wayne F
Department of Biochemistry and Molecular Genetics, Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.
Structure. 2017 Feb 7;25(2):295-304. doi: 10.1016/j.str.2016.12.007. Epub 2017 Jan 12.
Active in the aqueous cellular environment where a massive excess of water is perpetually present, enzymes that catalyze the transfer of an electrophile to a non-water nucleophile (transferases) require specific strategies to inhibit mechanistically related hydrolysis reactions. To identify principles that confer transferase versus hydrolase reaction specificity, we exploited two enzymes that use highly similar catalytic apparatuses to catalyze the transglycosylation (a transferase reaction) or hydrolysis of α-1,3-glucan linkages in the cyclic tetrasaccharide cycloalternan (CA). We show that substrate binding to non-catalytic domains and a conformationally stable active site promote CA transglycosylation, whereas a distinct pattern of active site conformational change is associated with CA hydrolysis. These findings defy the classic view of induced-fit conformational change and illustrate a mechanism by which a stable hydrophobic binding site can favor transferase activity and disfavor hydrolysis. Application of these principles could facilitate the rational reengineering of transferases with desired catalytic properties.
酶在水相细胞环境中发挥作用，而该环境中始终存在大量过剩的水。催化亲电试剂向非水亲核试剂转移的酶（转移酶）需要特定策略来抑制与机制相关的水解反应。为了确定赋予转移酶与水解酶反应特异性的原理，我们利用了两种酶，它们使用高度相似的催化装置来催化环状四糖环交替聚糖（CA）中α-1,3-葡聚糖键的转糖基化反应（一种转移酶反应）或水解反应。我们发现，底物与非催化结构域的结合以及构象稳定的活性位点促进了CA的转糖基化反应，而活性位点构象变化的独特模式与CA的水解相关。这些发现与诱导契合构象变化的经典观点相悖，并阐明了一种机制，即稳定的疏水结合位点可以促进转移酶活性而不利于水解。应用这些原理有助于对具有所需催化特性的转移酶进行合理的重新设计。
