Interaction of aromatic donor molecules with lactoperoxidase probed by optical difference spectra

Abstract

On the basis of optical difference spectra, lactoperoxidase (LPO) was shown to form a 1:1 complex with aromatic donor molecules: resorcinol, hydroquinone, phenol, p-cresol, guaiacol, aniline, and benzohydroxamic acid. As compared with horseradish peroxidase (HRP), the values of the dissociation constant, Kd, of LPO-donor complexes were found to be 4-720-fold larger and were not greatly changed in the presence of KCN and by changes in pH in the range 4-9. The apparent enthalpy and entropy of the binding reactions were found to be -13 kJ mol-1 and -29 J mol-1 K-1, respectively, somewhat smaller (in absolute value) than the corresponding values of HRP. The difference spectra of LPO-donor complexes resembled each other, in contrast to the case of HRP, and the bindings of the donors to LPO occurred in a competitive fashion between the donors. Incubation of LPO with phenylhydrazine and hydrogen peroxide markedly depressed donor binding, the intensity of the Soret band, and the catalytic activity, probably as the result of formation of meso-phenyl derivatives of the heme. These findings suggest that the binding of aromatic donors to LPO occurs at a specific site, probably near the heme edge, where the electron transfer in the peroxidase reaction may take place.

Mechanism-based inactivation of lacrimal-gland peroxidase by phenylhydrazine: a suicidal substrate to probe the active site

“…However, significant loss of absorption intensity was due to the lower absorption coefficient of the compound II form of the modified enzyme. A similar observation was also made in the case of modification of LPO with PH [25]. Although, because of the difficulty of splitting haem from LGP, it cannot be demonstrated at present that haem has been modified similarly to HRP [28], the comparable change in intensity without the shift of the Soret band indicates modification of the haem environment.…”

“…The difference spectra of the enzyme-donor complex versus enzyme [25,33] were recorded in a Shimadzu UV-2201 computerized spectrophotometer at 25p1 mC. Initially both the cuvettes were filled with 1n0 ml of the enzyme solution and the baseline was recorded.…”

“…Iproniazid causes severe liver damage [17], and hydrazine causes lupus erythematosus [18] and base pair mutation in bacterial and mammalian cells [19,20]. Hydrazine is already known to inactivate haemoglobin [21], catalase [22], horseradish peroxidase (HRP) [23], lactoperoxidase (LPO) [24,25], thyroid peroxidase [26] and monoamine oxidase [27]. Although the mechanism of inactivation of haemoglobin [21], catalase [22] and HRP [28] are available, very little is known about the detailed mechanism of inactivation of mammalian peroxidases.…”

Probing the Aromatic‐Donor‐Binding Site of Horseradish Peroxidase Using Site‐Directed Mutagenesis and the Suicide Substrate Phenylhydrazine

“…Sequence alignment of class 1 (prokaryote and yeast), 11 (fungal) and IT1 (higher plants) peroxidases clearly indicates that this insertion is unique to class 111 peroxidases (including HRP-C). CiP is able to form a stable 1 : 1 complex with benzhydroxamic acid, albeit with a high Kd (3.7 mM) with a typical type I binding spectrum [45]. An interesting possibility, therefore, is that the function of this additional structural element in Class I11 peroxidases may be to provide an additional component of a hydrophobic pocket involved in the binding/ retention of aromatic molecules.…”

Some Improved Versions of Methods for the Indirect Detection of Staphylococcal Protein A Gene Expressed in Escherichia coli

“…The filter was soaked in 10 mm phenylhydrazine supplemented with 1 mm hydrogen peroxide for 30 min. Phenylhydrazine is one of the most effective suicide substrates for peroxidase (3), and in this step, endogenous peroxidase was completely inactivated. …”