1 INTRODUCTION
Many enzymes in Enzyme Class 3 (Hydrolases) catalyze chemical group transfer to acceptors (e.g., alcohols) other than water. Such donor-to-acceptor transfer reactions are the basis for important applications of hydrolytic enzymes in organic synthesis (Adlercreutz, 2017; Giordano, Ribeiro, & Giordano, 2006; Kasche, 1986; Marsden, Mestrom, McMillan, & Hanefeld, 2020; Müller et al., in press; Seibel, Jördening, & Buchholz, 2006; Vera, Guerrero, Aburto, Cordova, & Illanes, 2020). Commercial-scale production of various chemicals and ingredients (e.g., oligosaccharides and glycosides (van Rantwijk, Woudenberg-van Oosterom, & Sheldon, 1999; Vera et al., 2020), esters and triglycerides (Adlercreutz, 2017; Mestrom, Claessen, & Hanefeld, 2019; Norjannah, Ong, Masjuki, Juan, & Chong, 2016), β-lactam antibiotics (Giordano et al., 2006; Kasche, 1986), phosphorylated nucleosides (Asano, Mihara, & Yamada, 1999; Ishikawa et al., 2002; Kato, Ooi, & Asano, 1999)) relies on hydrolase-catalyzed group transfer. Glycoside hydrolases (Adlercreutz, 2017; Seibel et al., 2006; Vera et al., 2020; Zeuner, Jers, Mikkelsen, & Meyer, 2014), lipases (Adlercreutz, 2017; Subileau et al., 2017), amidases (Bruggink, Roos, & de Vroom, 1998; Giordano et al., 2006; Gololobov et al., 1990; Sio & Quax, 2004; Sklyarenko, El’darov, Kurochkina, & Yarotsky, 2015) and phosphatases (Ishikawa et al., 2002; Tasnádi, Staśko, Ditrich, Hall, & Faber, 2020; Wildberger, Pfeiffer, Brecker, & Nidetzky, 2015) are industrial enzymes used in these synthetic processes. Mechanistically, the enzymatic reactions proceed in two catalytic steps via a covalent enzyme intermediate (Gololobov, Borisov, Belikov, & Švedas, 1988; Kasche, 1986; Marsden et al., 2020; Seibel et al., 2006; Zeuner et al., 2014). The first step involves group transfer from the donor substrate to an active-site nucleophile of the enzyme. The enzyme intermediate then reacts with the incoming acceptor (which is water in the canonical hydrolysis reaction) to release the synthetic product (Gololobov et al., 1988; Gololobov et al., 1990; Huber, Kurz, & Wallenfels, 1976; Kasche, 1986; Marsden et al., 2020; Vera et al., 2020; Youshko, Chilov, Shcherbakova, & Švedas, 2002). Synthesis in an aqueous environment operates in competition with hydrolysis (Adlercreutz, 2017). A minimal kinetic mechanism for enzymatic group transfer is shown in Figure 1. The mechanism implies that synthesis and hydrolysis product are formed in a ratio determined by enzyme selectivity but independent of the donor substrate used (Adlercreutz, 2017; Kasche, 1986; Marsden et al., 2020; Youshko et al., 2002).
The industrially used glycosylation of glycerol catalyzed by sucrose phosphorylase (SucP) (Bolivar, Luley-Goedl, Leitner, Sawangwan, & Nidetzky, 2017; Goedl, Sawangwan, Mueller, Schwarz, & Nidetzky, 2008; Luley-Goedl, Sawangwan, Mueller, Schwarz, & Nidetzky, 2010) represents a number of hydrolase-promoted transfer reactions for which the reaction scheme shown in Figure 1 is likely an oversimplification (e.g., (Fernandez-Lafuente, Rosell, & Guisan, 1998; Gololobov et al., 1988; Gololobov et al., 1990; Kasche, 1986; Terreni et al., 2005; Vera, Guerrero, Wilson, & Illanes, 2017; Youshko et al., 2002)). The SucP can use sucrose (1 ) or G1P (2 ) as donor for α-D-glucosyl-sn -glycerol (GG; 3 ) formation (Goedl, Sawangwan, et al., 2008; Goedl, Schwarz, Minani, & Nidetzky, 2007; Renirie, Pukin, van Lagen, & Franssen, 2010). However, the ratio between GG and glucose (hydrolysis product) is ∼1.5 – 2.5-fold higher for transfer from sucrose as compared to transfer from G1P, inconsistent with Figure 1 (Goedl, Sawangwan, et al., 2008). This study was conceptualized to achieve clarification. We considered that mechanistic insight into the donor substrate dependence of the enzymatic glucosyl transfer is fundamentally important, and can be practically significant, in a broad field of applied bio-catalysis using hydrolase enzymes for synthesis (Adlercreutz, 2017; Giordano et al., 2006; Kasche, 1986; Marsden et al., 2020; van Rantwijk et al., 1999).
We studied two SucP enzymes, from Leuconostoc mesenteroides(Lm SucP) (Goedl et al., 2007; Goedl, Schwarz, Mueller, Brecker, & Nidetzky, 2008; Luley-Goedl & Nidetzky, 2010) andBifidobacterium adolescentis (Ba SucP) (Mirza et al., 2006; van den Broek et al., 2004). Both enzymes have been well characterized previously (Franceus & Desmet, 2020; Goedl, Sawangwan, Wildberger, & Nidetzky, 2010; Goedl, Schwarz, et al., 2008), and the Lm SucP is used for commercial production of GG (Luley-Goedl et al., 2010). Guided by kinetic framework analysis developed in here, we show that in enzymatic reactions with G1P, hydrolysis takes place not only from the covalent glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and G1P which unlike E-Glc cannot be intercepted by glycerol. At G1P concentrations too low for substrate-bound E-Glc to form, the product ratio increases to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Besides explaining kinetic behavior of SucP not previously accounted for (Goedl, Sawangwan, et al., 2008; Renirie, Pukin, van Lagen, & Franssen, 2010), these results are useful to facilitate model-based optimization of GG synthesis. In addition, our findings expand the theoretical basis for analyzing kinetically analogous group transfer reactions by hydrolytic enzymes (e.g. (Adlercreutz, 2017; Ishikawa et al., 2002; Kasche, 1986)).