Role of Tyrosine-103 in Myoglobin Peroxidase Activity: Kinetic and Steady-State Studies on the Reaction of Wild-Type and Variant Recombinant Human Myoglobins with H2O2
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- 作者：Paul K. Witting ; A. Grant Mauk ; and Peter A. Lay
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：September 24, 2002
- 年：2002
- 卷：41
- 期：38
- 页码：11495 - 11503
- 全文大小：110K
- 年卷期：v.41,no.38(September 24, 2002)
- ISSN：1520-4995
文摘
Myoglobin (Mb) catalyzes a range of oxidation reactions in the presence of hydrogen peroxide(H2O2) through a peroxidase-like cycle. C110A and Y103F variants of human Mb have been constructedto assess the effects of removing electron-rich oxidizable amino acids from the protein on the peroxidaseactivity of Mb: a point mutation at W14 failed to yield a viable protein. Point mutations at C110 andY103 did not result in significant changes to structural elements of the heme pocket, as judged by low-temperature electron paramagnetic spectroscopy (EPR) studies on the ground-state ferric proteins. However,compared to the native protein, the yield of globin radical (globin
) was significantly decreased for theY103F but not the C110A variant Mb upon reaction of the respective proteins with H2O2. In contrastwith our expectation that inhibiting pathways of intramolecular electron transfer may lead to enhancedMb peroxidase activity, mutation of Y103 marginally decreased the rate constant for reaction of Mb withH2O2 (1.4-fold) as judged by stopped-flow kinetic analyses. Consistent with this decrease in rate constant,steady-state analyses of Y103F Mb-derived thioanisole sulfoxidation indicated decreased Vmax and increasedKm relative to the wild-type control. Additionally, thioanisole sulfoxidation proceeded with lower stereoselectivity, suggesting that Y103 plays a significant role in substrate binding and orientation in the hemepocket of Mb. Together, these results show that electron transfer within the globin portion of the proteinis an important modulator of its stability and catalytic activity. Furthermore, the hydrogen-bonding networkinvolving the residues that line the heme pocket of Mb is crucial to both efficient peroxidase activity andstereospecificity.
) was significantly decreased for theY103F but not the C110A variant Mb upon reaction of the respective proteins with H2O2. In contrastwith our expectation that inhibiting pathways of intramolecular electron transfer may lead to enhancedMb peroxidase activity, mutation of Y103 marginally decreased the rate constant for reaction of Mb withH2O2 (1.4-fold) as judged by stopped-flow kinetic analyses. Consistent with this decrease in rate constant,steady-state analyses of Y103F Mb-derived thioanisole sulfoxidation indicated decreased Vmax and increasedKm relative to the wild-type control. Additionally, thioanisole sulfoxidation proceeded with lower stereoselectivity, suggesting that Y103 plays a significant role in substrate binding and orientation in the hemepocket of Mb. Together, these results show that electron transfer within the globin portion of the proteinis an important modulator of its stability and catalytic activity. Furthermore, the hydrogen-bonding networkinvolving the residues that line the heme pocket of Mb is crucial to both efficient peroxidase activity andstereospecificity.