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.