A structural study of ribokinase from Escherichia coli
Abstract: Ribokinase phosphorylates the abundant sugar ribose, using ATP in the presence of magnesium. The phosphorylation is the first reaction in ribose metabolism and the product, ribose-5-phosphate, can enter the pentose phosphate pathway and glycolysis. It can also be used in the synthesis of histidine and purines. Ribokinase belongs to the ribokinase family which includes various carbohydrate kinases from both prokaryotes and eukaryotes. No member's three-dimensional structure has previously been determined.In this thesis the threedimensional structure of ribokinase from Escherichia coli, determined by X-ray crystallography, is presented. The enzyme is a dimer and each subunit consists of a large a/b domain and a small b-sheet domain. The two b-sheet domains interact and form a b-clasp at the dimer interface. The two active sites are found in a cleft between the b-clasp and the a/b domains.Further structural studies on the enzyme in the apo form and in binary and ternary complexes show that the enzyme has both open and closed states. Ribose induces the conformational change into the closed form. The small b-sheet domain covers the ribose-binding site as a lid and produces a good micro environment for the phosphorylation reaction.The ordered reaction is thought to involve a catalytic acid (Asp255) and go through an in-line mechanism where the ribose O5' is the nucleophile attacking the g-phosphate of the ATP. The penta-covalent transition state can be stabilised by a magnesium ion, an anion hole and a lysine side chain. Preliminary kinetic studies have determined the KM for ribose to be 0.2 mM. These results may be applicable to other members of the family.
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