The binding behavior of a competitive inhibitor, N-phenylbenzhydroxamic acid
(BHA) against horseradish peroxidase (HRP) was studied in order to understand and
predict the interaction mechanism of hydrogen donors with the enzyme. The
dissociation constants of the complexes of HRP-BHA, HRP-donor and HRP-BHA-azide were estimated at specified conditions by difference spectroscopy. The binding site of BHA and that of hydrogen donor were also detected by spectroscopic titration of HRP in the presence of BHA and specific amino acid modifiers. A histidine binding site with pK = 6.40 was detected for the binding of BHA. On the other hand, activity-pH measurements showed that the maximum interaction of BHA with HRP occurs at a minimum point of activity at pH=6.40. The catalytic behavior of His 42 in the kinetic mechanism of enzyme action was also discussed