STUDIES ON L-AMINO-ACID OXIDASE .I. EFFECTS OF PH AND COMPETITIVE INHIBITORS

1. 1. Aromatic carboxylates are found to be competitive inhibitors of l-amino-acids oxidase (l-amino-acid:O2 oxidoreductase (deaminating), EC 1.4.3.2). The inhibitor constants for a number of ring substituted benzoates and some other compounds have been determined. They do not follow a linear Hammett relationship. 2. 2. l-Amino-acid oxidase reacts immediately with these inhibitors to form spectrally detectable enzyme-inhibitor complexes. Almost all inhibitors cause a blue shift of either one or both visible absorption bands of the flavoprotein and have influences on the extinction coefficients of their absorption maxima. These effects may be explained by indirect changes in the environment of the isoalloxazine rather than by direct complex formation of the inhibitor with this ring system. The enzyme complex with o-aminobenzoate gives an additional broad band, extending from 540 to 750 nm. o-Mercaptobenzoate causes a red shift of the long-wavelength band of the enzyme, immediately followed by a slow and incomplete reduction of the enzyme-bound FAD. 3. 3. Kinetically determined inhibition constants are in good agreement with the dissociation constants of the enzyme-inhibitor complexes, determined by titraton of the enzyme with the inhibitor at the same pH and temperature. 4. 4. The dissociation constants, thus determined, were found to be dependent on the pH of the reaction mixture. For the l-amino-acid oxidase-o-aminobenzoate complex, the dissociation constant had a minimum value of 0.24 mM at pH 8.6. From curves relating pH to absorbance differences, a group in the free enzyme was found with an apparent pKa value of 7.80, which participates in the binding of the inhibitor. 5. 5. The spectrum of the free enzyme is also dependent on the pH. An instantaneous and reversible blue shift is observed when going from pH 5.0 to pH 7.0, with a midpoint near pH 6.1. As a result, the unrsolved 460-nm band at pH 5.0 becomes slightly resolved at higher pH. Above pH 8.2 an additional slow and apparently irreversible blue shift occurs, which is also found on inactivation of the enzyme in the presence of 0.1 M phosphate, at lower pH and higher temperature, as described previously. © 1968.