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Abstract:
Phosphate plays numerous indispensable biochemical roles in all living organisms. Many enzymes act on the phosphorylated form of substrates rather than on the nonphosphorylated form. Usually, the phosphate group does not directly participate in catalysis but is required for substrate binding and recognition. Also, it was suggested that several enzymes utilize the binding energy of the phosphoanion for enzyme activation. However, potential roles of the substrate phosphate group in other stages of enzyme catalysis (e.g., intermediate stabilization or destabilization) have not been documented. The enzyme transketolase (TK), which is involved in the biosynthesis of nucleic acids and aromatic amino acids, is a key enzyme in the pentose phosphate pathway and acts on phosphoketose and phosphoaldose substrates. Previous studies of TK with non-native, nonphosphorylated sugars had revealed an anchoring role for the substrate phosphate group. In this study, we provide new evidence of several catalytic functions of the substrate phosphate group. First, it facilitates the formation of the distorted, high-energy substrate-ThDP conjugate, which is critical for transketolase catalysis, by means of reactant-state destabilization. Second, it prevents the accumulation of the reactive Breslow enamine intermediate and thus abortive side reactions such as protonation or oxygenation, suggesting a gated acceptor exchange mechanism. Third, the binding of the exogenous phosphite anion multifold enhances the reactivity on nonphosphorylated substrates. Altogether, our studies have delineated several previously unrecognized roles for the substrate phosphate group in transketolase catalysis, thus underscoring the seminal statement of “why nature chose phosphates” that Westheimer proposed in 1987.
