4 DISCUSSION
Using mechanism-based kinetic analysis, we clarify in this study the peculiar behavior of sucrose phosphorylase, that enzyme selectivity for product formation via glycosylation of glycerol and hydrolysis depends on the type and concentration of the glucosyl donor substrate used (Goedl, Sawangwan, et al., 2008; Goedl, Schwarz, Minani, & Nidetzky, 2007; Renirie, Pukin, van Lagen, & Franssen, 2010). We present strong evidence for an unanticipated kinetic complex of E-Glc with G1P that can hydrolyze to regenerate the free enzyme upon release of glucose and G1P, but is unable to form the transglycosylation product GG. The molecular interactions involved in the proposed complex are not known in detail. However, suffice it to emphasize that the (acceptor) binding pocket of the E-Glc form of Ba SucP is wide and flexible (Mirza et al., 2006; Sprogøe et al., 2004) to accommodate a range of bulky acceptors (Dirks-Hofmeister, Verhaeghe, De Winter, & Desmet, 2015; Goedl et al., 2010; Seibel et al., 2006), including glucose in multiple orientations (Beerens et al., 2017; Kraus, Görl, Timm, & Seibel, 2016; Verhaeghe et al., 2016). The kinetic significance of the (E-Glc··G1P) complex, in terms of the amount formed at steady state and the rate of breakdown, have strong implications on canonical (apparent) enzyme kinetic parameters obtained from standard experiments (e.g.\({}^{\text{app}}V_{X}\), \({}^{\text{app}}K\), TC; see Table 1). The deepened mechanistic understanding obtained from this research enables causes to be related quantitatively to effects and so unifies conflicting evidence from different studies of sucrose phosphorylase (Goedl, Sawangwan, et al., 2008; Renirie, Pukin, van Lagen, & Franssen, 2010) into a coherent whole. The current study comprises two further elements of broad importance. Firstly, the kinetic framework analysis (Figure 2 and the associated algebra in the Supporting Information Table S1) provides extended basis for the kinetic evaluation of other hydrolytic enzymes that catalyze chemical group transfer reactions (e.g., glycosyl, phosphoryl, acyl). As discussed later, there have been numerous studies of such enzymes over decades, but a comprehensive analysis of the different kinetic scenarios surrounding the covalent enzyme intermediate has been worked out only for selected transformations, in particular those of amidases/acyltransferases in β-lactam synthesis (Gololobov et al., 1988; 1990; Schroën et al., 2001; Youshko et al., 2002). Disagreement with the base mechanism M1 has been noted in several instances however (Fernandez-Lafuente et al., 1998; Kasche, 1986; Terreni et al., 2005; Vera et al., 2017). Secondly, the kinetic analysis of sucrose phosphorylase provides essential basis for design and optimization of the synthetic reaction for GG production, as follows.
Analyzing the reaction with sucrose and glycerol (Table 1), one recognizes that the kinetic fitness is higher for Ba SucP thanLm SucP. The higher transfer coefficient TC forBa SucP derives mostly from a hydrolysis rate 3-fold (= 2.68/0.88; Table 1) lower than for Lm SucP. Using 20 mM sucrose as the donor, one could do with a 1.74-fold (= 8.2/4.7; Table 1) lower glycerol concentration to achieve the same product selectivity GG/Glc. To have a selectivity of 10, one would need 1.2 M glycerol using Ba SucP.
The reaction with G1P has been challenging to use for GG synthesis. Thermodynamically, G1P is less preferred for glycosylation of glycerol than sucrose (Goedl et al., 2007). However, as has been shown for reactions of other phosphorylases, the reaction of G1P can be made quasi-irreversible through in situ product removal, using precipitation of the released phosphate in the presence of Mg2+(Zhong, Luley-Goedl, & Nidetzky, 2019). Separation of GG from phosphate is readily achieved whereas separation from fructose is more difficult (Kruschitz & Nidetzky, 2020). Therefore, G1P remains an option for donor substrate. Kinetic models for Ba SucP and Lm SucP enable rigorous “window of operation” analysis to identify conditions suitable for the synthesis. We scanned [G1P] in the range 5 – 70 mM (Lm SucP) and 50 – 700 mM (Ba SucP) at glycerol concentrations varying between 0.85 and 2.0 M. We calculated the\(v_{X}\) and \(\frac{v_{X}}{v_{H}}\) corresponding to each condition and defined the operational window according to the requirement that\(v_{X}\) and \(\frac{v_{X}}{v_{H}}\) each be at least 80% of the maximum value. Within the window of operation (Supporting Information Figure S3), we identified the optimum point as involving the smallest [GOH]. For Ba SucP, we get [G1P] = 650 mM; [GOH] = 1.55 M; \(v_{X}\) = 20.2 s-1. For Lm SucP, we get [G1P] = 15 mM; [GOH] = 1.83 M; \(v_{X}\) = 17.3 s-1. Ba SucP is clearly preferred. UsingLm SucP, synthesis of GG in concentrations exceeding 15 mM should not be performed in batch. A fed-batch mode of reaction could be used in which 15 mM donor substrate is added according to progress of the reaction. However, a fed-batch process would involve substantially higher complexity due to the reaction control required. Evidently, it would be a highly laborious task to (try to) identify the respective windows of operation for Ba SucP and Lm SucP from experiments.
Although the underlying reasons may differ among different enzymes, the effect is common: it is true for numerous hydrolases that the efficiency of chemical group transfer is dependent on the type of donor substrate used (Fernandez-Lafuente et al., 1998; Kasche, 1986; Terreni et al., 2005; van Rantwijk et al., 1999; Vera et al., 2017). Glycoside hydrolases often can use a variety of donor substrates for hydrolysis (van Rantwijk et al., 1999; Vera et al., 2017; Zeuner et al., 2014). However, as shown for the β-glycosidase CelB from Pyrococcus furiosus , glycosyl transfer to acceptors from lactose is substantially less efficient when lactose is used compared to nitrophenyl-β-D-galactoside (Petzelbauer, Reiter, Splechtna, Kosma, & Nidetzky, 2000). Like most acid phosphatases, the G1P phosphatases fromE. coli accepts a broad variety of donors for hydrolysis (e.g., nitrophenyl-phosphate, pyrophosphate) but transfer to acceptors is efficient only when G1P is used (Wildberger, Pfeiffer, Brecker, & Nidetzky, 2015; Wildberger, Pfeiffer, Brecker, Rechberger, et al., 2015). Even close homologues of G1P (e.g., α-D-mannose-1-phosphate) fail to elicit efficient transfer (unpublished results, 2020). The influence of the donor substrate is considerably more pronounced than in sucrose phosphorylase. Other phosphatases behave similarly (Han & Coleman, 1995; Tasnádi et al., 2020; van Herk, Hartog, van der Burg, & Wever, 2005). Similar behavior has been noted for select esterases/lipases and proteases/N-acylases (Adlercreutz, 2017; Marsden et al., 2020; Müller et al., in press). Extending previous studies, notably those on amidases and acyltransferases (Gololobov et al., 1988; Gololobov et al., 1990; Youshko et al., 2002), the kinetic framework analysis reported here could be useful in the study of these enzymes and group transfer reactions catalyzed by them.