THE MANGANESE BINDING-SITE OF MANGANESE PEROXIDASE - CHARACTERIZATION OF AN ASP179ASN SITE-DIRECTED MUTANT PROTEIN

A site-directed mutant, D179N, in the gene encoding Phanerochaete chrysosporium manganese peroxidase isozyme 1 (mnp1), was created by overlap extension, using polymerase chain reaction. The mutant gene was expressed in P. chrysosporium under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidase (MnP) was purified, and its spectra and MW were very similar to those of the wild-type enzyme. Steady-state kinetic analysis of MnP D179N revealed that the K-m for the substrate Mn-II was similar to 50-fold greater than the corresponding K-m for the wildtype recombinant enzyme (3.7 mM versus similar to 70 mu M). Likewise, the k(cat) value for Mn-II oxidation of the mutant protein was only 1/265 of that for the wild-type enzyme. By comparison, the apparent K-m for H2O2 of MnP D179N was similar to the corresponding value of the wild-type MnP. The first-order rate constant for MnP D179N compound II reduction by Mn-II was approximately 1/200 of that for the wildtype enzyme. The equilibrium dissociation constant (K-D) for MnP D179N compound II reduction by Mn-II was similar to 100-fold greater than the KD for the wild-type compound II. In contrast, the second-order rate constant for p-cresol reduction of the mutant compound II was similar to that of the wild-type enzyme. These results also suggest that the mutation affects the binding of Mn-II to the enzyme and, consequently, the rate of compound II reduction by Mn-II. In contrast, the mutation apparently does not have a significant effect on H2O2 cleavage during compound I formation or on p-cresol reduction of compound II. The results strongly suggest that Asp179 is one of the acidic amino acid ligands in the Mn-II binding site of MnP.