Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

Abstract

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 (k cat = 84 s–1) and reverse direction (k cat = 22 s–1) of reaction at 30 °C. Enzyme–substrate association is driven by entropy (TΔS b ≥ +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 (k non), ΔΔG ⧧ = ΔG non ⧧ – ΔG enzyme ⧧ = +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 (k cat/k non) is ∼1012-fold and similar for reactions of sucrose and Glc1P. The 103-fold lower reactivity (k cat/K m) 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.

Limits of Non‐invasive Enzymatic Activation by Local Temperature Control

“…This enzyme was recombinantly expressed and purified (GeneBank: AAO84039; Uniprot: Q84BY1) via a polyhistidine tag at N‐terminus (His‐tag) with high specific activity of 79 (± 20) U mg −1 at 30 °C. [ 40,41 ] The enzyme catalyzes reversible conversion of sucrose and inorganic phosphate to α‐D‐glucose 1‐phosphate (Glc1P) and fructose (Figure S3, Supporting Information). This reaction is of interest in context of different multienzyme cascade transformations, to provide Glc1P as reactive donor substrate of synthetic glucosylations [ 42–45 ] (Figure S3, Supporting Information) and to scavenge the phosphate released in kinase‐phosphatase reaction sequences (for, e.g., islatravir pathway).…”

“…As a model enzyme we choose sucrose phosphorylase, which is known to show substantial thermal activation (Δ H = 72 kJ mol −1 ). [ 40 ] We investigated its activity increase when immobilized on MNPs under the influence of AMF, and tried to relate this activation to the increase in local and bulk temperature (Figure 1). As the existence of controlled temperature differences on the nano‐ or microscale would allow selective activation of two or more enzymes, for example in a multienzymatic cascade, we explored the conditions under which this could be realistically achievable.…”

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development

“…Parameter estimates (Table S3) and other constraints (Table S6) are generally found at intermediate. However, there is good evidence that the halfreaction with Suc (Figure 1) is rate-limiting (Vyas & Nidetzky, 2023).…”