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.