Sucrose Phosphorylase - Structural Implications

Structural Implications

The structure of sucrose phosphorylase has been identified in numerous experiments. The enzyme consists of four major domains, namely A, B, B’, and C. Domains A, B’ and C exist as dimers around the active site (Sprogoe et al. 2004). The size of the enzyme, as determined by sedimentation centrifugation, was found to be 55 KDa, consisting of 488 amino acids (Koga et al. 1991). The active has been shown to contain two binding sites, one designated a water site where hydroxylic molecules such as 1,2-cyclohexanediol and ethylene glycol may bind, and another designated as the acceptor site where the sugar molecule binds. Though the function of the water site has not been completely elucidated, the enzyme’s stability in aqueous solutions indicates that the water site may be involved in hydrolysis of the glycosidic bond.

The acceptor site is surrounded by three active residues that have been found to be essential in enzymatic activity. Using specific mutagenic assays, Asp-192 was found to be the catalytic nucleophile of the enzyme, “attacking C-1 of the glucosyl moiety of sucrose” (Schwarz and Nidetzky 2006). In fact, in vitro manipulation has shown that D-xylose, L-sorbose, and L-arabinose can replace fructose as the glucosyl acceptor (Mieyal, Simon, and Abeles 1972). The only requirement of the acceptor molecule is that the hydroxyl group on the C-3 be cis-disposed to the oxygen atom of the glycosidic bond. Glu-232 acts as the Bronsted acid-base catalyst, donating a proton to the displaced hydroxyl group on C-1 of the glucoside (Schwarz, Brecker, and Nidetzky 2007).

The most significant residue in the enzymatic activity, however, is Asp-295 (Mueller and Nidetzky 2007). Upon cleavage of the fructofuranosyl moiety from sucrose, the resultant glucose forms a covalent intermediate with the enzyme. The carboxylate side chain of Asp-295 hydrogen bonds with the hydroxyl groups at C-2 and C-3 of the glucosyl residue (Mueller and Nidetzky 2007). This interaction is maximized during the transition state of this covalent complex, lending support to the ping-pong mechanism. Finally, phosphorylation of the glucosyl residue at C-1 forms a transient positive charge on the glucosyl carbon, promoting breakage of the ester bond between Asp-192 and the sugar residue (Schwarz and Nidetzky 2006). Cleavage yields the product, α-D-glucose-1-phosphate.