Reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with N-methyl-5-hydroxynicotinic acid: studies on the mode of binding, and protonation status of the substrate.

Titrations of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate MHPC identified the MHPC species bound to the enzyme as the tripolar ionic species. This result was supported by studies of the binding to the enzyme of N-methyl-5-hydroxynicotinic acid (NMHN), an MHPC analog existing only in the tripolar ionic form. The Kd is 55 microM compared to a Kd of 9.2 microM for MHPC and 5.2 microM for 5-hydroxynicotinic acid. Kinetics studies of the binding of NMHN to MHPC oxygenase show that its binding, like that for MHPC and for 5HN, is also a two-step process. Since NMHN never exists as an anionic form, neither of the observed steps is due to the binding of an anionic species as an intermediate step. Investigations of the reduction and oxygenation half reactions demonstrate that the mechanism of catalysis with NMHN is basically the same as with MHPC or with 5-hydroxynicotinic acid. Product analysis from reactions using NMHN, a compound that possesses positive charge on the nitrogen atom, indicates that the product of NMHN is an aliphatic compound, similar to the products derived from MHPC and from another substrate analog, 5-hydroxynicotinic acid. These results indicate that the nitrogen atom of the substrate is invariably protonated during the catalytic reaction.