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 Km for the substrate MnII was approximately 50-fold greater than the corresponding Km for the wild-type recombinant enzyme (3.7 mM versus approximately 70 microM). Likewise, the kcat value for MnII oxidation of the mutant protein was only 1/265 of that for the wild-type enzyme. By comparison, the apparent Km 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 MnII was approximately 1/200 of that for the wild-type enzyme. The equilibrium dissociation constant (KD) for MnP D179N compound II reduction by MnII was approximately 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 MnII to the enzyme and, consequently, the rate of compound II reduction by MnII. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Kusters-van Someren
M
M
Department of Chemistry, Oregon Graduate Institute of Science & Technology, Portland 97291-1000, USA.
Kishi
K
K
Lundell
T
T
Gold
M H
MH
D001665:Binding Sites; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011124:Polyporaceae / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D011994:Recombinant Proteins; D008345:Manganese; D006861:Hydrogen Peroxide; D010544:Peroxidases; C051129:manganese peroxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases