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 (∼1014M–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.