Thermodynamics and Reduction Kinetics Properties of 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase

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Abstract
The investigation by absorbance and fluorescence rapid reaction spectrophotometry of the binding of the substrate MHPC (2-methyl-3-hydroxypyridine-5-carboxylic acid) or the substrate analog 5HN (5-hydroxynicotinic acid) to the flavoprotein MHPCO (2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase) shows that the binding proceeds in two steps. An enzyme−substrate complex initially formed is followed by a ligand-induced isomerization. This binding process is required for efficient reduction of the enzyme-bound flavin, as evidenced by the fact that MHPCO−substrate complexes can be reduced by NADH much faster than the enzyme alone. Since redox potential values of MHPCO and MHPCO−substrate complexes are the same, steric factors, such as the relative orientation of MHPC to the enzyme-bound flavin, are important for efficient hydride transfer to occur.