Ligand-induced tightening of oligomer structure: analyses of denaturation pathways reveal how oxyanions stabilise a bacterial a-glucan phosphorylase Richard Griessler Sabato D'Auria Fabio Tanfani Bernd Nidetzky* *Corresponding Author Keywords: phosphorylase, dimer stability, subunit interactions
Glucan phosphorylases are dimeric proteins composed of identical, approx. 90-kDa subunits and contain 1 mol pyridoxal 5'-phosphate per mol subunit as essential active-site cofactor. In the presence of phosphate, phosphorylases degrade starch or other a-glucans into the activated glucosyl donor a-D-glucose 1-phosphate which in turn serves as substrate of several enzymes involved in carbohydrate synthesis. Therefore, they have potential for use in enzymatic synthesis of value-added carbohydrates derived from starch. We are interested to unravel the mechanism by which oxyanions such as phosphate or sulfate bring about an extremely efficient stabilisation of the starch phosphorylase from the soil bacterium Corynebacterium callunae against denaturation by temperature or chaotropic agents such as urea. By binding to a protein site remote from the active site, dianionic phosphate induces formation of a more compact structure of phosphorylase, manifested by (i) an increase by about 5% in the relative composition of a-helical secondary structure, (ii) reduced 1H/2H exchange, and (iii) protection of cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by dissociation of protein subunits (manifested by the release into solution of pyridoxal 5'-phosphate), and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display substantial melting of secondary structure. Presumably by bridging the dimer interface, oxyanions stabilise the dimer structure and thereby preserve a functional active site, and stabilise the covalent protein-cofactor linkage. Since structure/function relationships of regulated and nonregulated a-glucan phosphorylases are known at high resolution, the results reported in this paper could have implications for the design of additional stabilising interactions in oligomeric proteins based on the binding of moderate-affinity oxyanion ligands. The extra stability conferred by the ligation of starch phosphorylase with phosphate is expressed as a dramatic shift of the entire denaturation pathway to a ˜ 20 °C higher value on the temperature scale or a 4 M higher value on the denaturant concentration scale. |
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