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Transient State Analysis of Porcine Dihydropyrimidine Dehydrogenase Reveals Reductive Activation by NADPH

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Transient State Analysis of Porcine Dihydropyrimidine Dehydrogenase Reveals Reductive Activation by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O O X X HN HN + NADPH + NADP+ O N O N H H pyrimidine dihydropyrimidine X = H, CH3, F, I Scheme 1: The chemistry catalyzed by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