Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

From its structure and mechanism, sucrose phosphorylase is a specialized glycoside hydrolase that uses phosphate ions instead of water as the nucleophile of the reaction. Unlike the hydrolysis reaction, the phosphate reaction is readily reversible and, here, this has enabled the study of temperature effects on kinetic parameters to map the energetic profile of the complete catalytic process via a covalent glycosyl enzyme intermediate. Enzyme glycosylation from sucrose and α-glucose 1-phosphate (Glc1P) is rate-limiting in the forward (kcat = 84 s–1) and reverse direction (kcat = 22 s–1) of reaction at 30 °C. Enzyme–substrate association is driven by entropy (TΔSb ≥ +23 kJ/mol), likely arising from enzyme desolvation at the binding site for the leaving group. Approach from the ES complex to the transition state involves uptake of heat (ΔH⧧ = 72 ± 5.2 kJ/mol) with little further change in entropy. The free energy barrier for the enzyme-catalyzed glycoside bond cleavage in the substrate is much lower than that for the non-enzymatic reaction (knon), ΔΔG⧧ = ΔGnon⧧ – ΔGenzyme⧧ = +72 kJ/mol; sucrose. This ΔΔG⧧, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (∼1014 M–1), is almost entirely enthalpic in origin. The enzymatic rate acceleration (kcat/knon) is ∼1012-fold and similar for reactions of sucrose and Glc1P. The 103-fold lower reactivity (kcat/Km) of glycerol than fructose in enzyme deglycosylation reflects major losses in the activation entropy, suggesting a role of nucleophile/leaving group recognition by the enzyme in inducing the active-site preorganization required for optimum transition state stabilization by enthalpic forces.


S1.1. Recombinant gene expression
Escherichia coli BL21-Gold (DE3) cells harboring recombinant plasmid encoding sucrose phosphorylase (reported in literature) 1 were cultivated in baffled shaken flasks (1 L) at 37 °C using 500 mL LB media (5 g L −1 NaCl, 5 g L −1 yeast extract, 10 g L −1 peptone) supplemented with 115 g L −1 ampicillin. The culture flasks were agitated at 110 rpm using an incubator shaker, CERTOMAT BS-1 (Sartorius, Germany). Gene expression was induced by the addition of 250 μM IPTG at OD600 0.8 -1.0 and the cells were incubated overnight at 25°C. Cells harvested by centrifugation at 6,000 g (4°C) for 20 min were resuspended in 50 mM potassium phosphate buffer, pH 7.0 to a concentration of 100 g L −1 . The cells were disrupted by ultrasonication using Sonic Dismembrator Model 505 (Fisher Scientific, USA) at 60% amplitude comprising of three cycles of 6 min (2 s pulse on and 4 s pulse off). Cell debris were removed by ultracentrifugation at 30,000 rcf (4°C) for 45 min. The resulting supernatant was filtered using 0.45 μm cellulose acetate filter (Sartorius, Germany) and was further used for purification.

S1.2. Protein purification
The purification steps were carried out with an Äkta explorer system (GE Healthcare, Germany) at 4°C assisted with protein detection at 280 nm. Volume of 50 ml of cell free extract (50 mg protein/mL) was applied to nickel sepharose high performance affinity resin packed HisTrap column (16  25 mm, 5 mL; GE Healthcare, Germany) equilibrated with 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M sodium chloride and 50 mM imidazole. All buffer solutions used were filtered using 0.45 μm cellulose-acetate filter (prior to use). Gradient elution was achieved at a constant flow rate of 2 mL min −1 using 20 mM sodium phosphate buffer pH 7.0, containing 0.5 M sodium chloride and 0.5 M imidazole. The enzyme was eluted at 100 − 150 mM imidazole and S5 was concentrated by ultrafiltration using Vivaspin Turbo 15 tubes (Sartorius, Germany) with 30 kDa molecular cut off. Buffer solution was changed to 50 mM potassium phosphate buffer (pH 7.0). The presence of purified recombinant sucrose phosphorylase was confirmed by running a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in a precast 4 -12 % gradient gel on an Xcell surelock system (both from Invitrogen, Austria). Bands were visualized by staining the gel using Coomassie Brilliant Blue dye based on molecular weights. Aliquots of 250 μL of purified enzyme (50 mg/mL) supplemented with 5 % glycerol were stored at −20°C for further use (temperature dependent reactions and immobilization).

S1.3. Measurement of analyte
Time dependent samples from temperature-controlled reactions were analyzed for product(s) based on the conversion assisted by sucrose phosphorylase (Figure 1a). Procedures to analyze different compounds entailed in this study are described as following.

S1.3.1. Glc1P analysis
-Glucose 1-phosphate (Glc1P) concentrations were determined using a colorimetric assay where the formation of NADH was detected at 340 nm using 3.1 mM NAD + , 2.7 U glucose-6phosphate dehydrogenase (G6PDH) and 3.1 U phosphoglucomutase in 50 mM Tris/HCl buffer (pH 7.7) with 10 mM magnesium chloride and 10 μM α-D-glucose 1,6-bisphosphate. Reagent to sample (or standard) volume was kept as 114 µL to 80 µL. And the samples were analyzed against a standard curve by reading absorbances on a plate reader at 340 nm.

S1.3.2. Phosphate analysis
Phosphate analyses were performed using a colorimetric assay 2 . Here, molybdate reagent consisting of 15 mM ammonium molybdate and 100 mM zinc acetate at pH 5.0 was mixed with 10% (m/v) L-ascorbic acid in a ratio of 4:1 respectively. 20 µL of sample was dispensed to this S6 reagent (140 µL) and incubated for 15 mins at 30C. Reduction of phosphomolybdate complex was read at 850 nm in a microplate reader. And the quantification was based on a standard curve.

S1.3.3. Glucose and fructose analysis
The analysis of glucose and fructose was based on a coupled enzymatic assay using hexokinase (HK) and G6PDH 3 . The measurement was performed using a commercial assay kit (K-SURFG, Megazyme, Ireland). The commercial buffer was mixed with NADP + /ATP and HK/G6PDH to prepare the reagent (8:8:1 by volume respectively) 4 . This reagent (140 µL) was added to samples (80 µL) and the end point absorbances were read at 340 nm (after 20 mins) to estimate glucose concentration 4 . After reading the absorbances for glucose in a microplate reader, an additional 20 µL of 40 times diluted phosphoglucose isomerase solution was added to quantify fructose concentration 4 . Quantification of either analytes was based on a standard plot.

S1.4. Estimation of acid hydrolysis rate of sucrose
The uncatalyzed rate of acid hydrolysis of sucrose was estimated from literature 5 where pH dependent acid hydrolysis rate (kacid) is expressed in terms of activation energy (Ea = 99 kJ mol −1 ) and its associated rate constant (ln k0 = 35.6) 5 . The relationship was used (Equation 8 described in the paper) 5 for the estimation. The rate of acid hydrolysis of sucrose was calculated as 4  10 −4 s −1 , while at pH 7.0 (kchem = kacid 10 −pH ), the rate was estimated as 4  10 −11 s −1 .

S1.5. Estimation of acid hydrolysis rate of Glc1P
The uncatalyzed rate of Glc1P hydrolysis via C1-O bond cleavage was estimated from literature 6 . For comparative evaluation with sucrose phosphorylase catalyzed reactions, it's crucial that uncatalyzed hydrolysis rate is determined at pH 7.0 and 30C. The pH dependent logarithmic plot of rates (at 82C) 6 in the range of pH 1.23 -3.33 ( Figure S10) denotes C1-O bond cleavage (in acidic region). To estimate the rate at pH 7.0, extrapolation was performed ( Figure S10) and the S7 rate was calculated at 82C (5.7  10 −9 s −1 ). Following it, the rate at 30C was estimated using the activation energy (temperature dependence). The calculation was done by equating the slope i.e.  Table S1 for detailed reaction composition. And the associated derived parameters from the fits are presented in Table 1.  Table S1 for detailed reaction composition. And the associated derived parameters from the fits are presented in Table 1.  Table S1 for detailed reaction composition. And the associated derived parameters from the fits are presented in Table 1.  Table S1 for detailed reaction composition. And the associated derived parameters from the fit are presented in Table 1.  Table S1. Compiled conditions for temperature-based characterization of respective reactions assisted by sucrose phosphorylase at pH 7.0 ( Figure 1).

Reaction
To  S22 Table S2. Energy levels plotted in the reaction coordinate diagram i.e., Figure 4.
a Values were estimated at 303 K. b knon was used for uncatalyzed reactions.