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Journal article
Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase
Biochemistry (Easton), v 45(19), pp 5985-5992
16 May 2006
PMID: 16681370
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M(-)(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the anionic form of the amino acid, the results indicate that the pK(a) of enzyme-bound sarcosine must be considerably lower than the free amino acid (pK(a) = 10.0). No redox intermediate is detectable during sarcosine oxidation, as judged by the isosbestic spectral course observed for conversion of E-FAD(ox).sarcosine to reduced enzyme at 25 or 5 degrees C. The limiting rate of the reductive half-reaction at 25 degrees C (140 +/- 3 s(-)(1)) is slightly faster than turnover (117 +/- 3 s(-)(1)). The kinetics of formation of the Michaelis charge transfer complex can be directly monitored at 5 degrees C where the reduction rate is 4.5-fold slower and complex stability is increased 2-fold. The observed rate of complex formation exhibits a hyperbolic dependence on sarcosine concentration with a finite Y-intercept, consistent with a mechanism involving formation of an initial complex followed by isomerization to yield a more stable complex. Similar results are obtained for charge transfer complex formation with methylthioacetate. The observed kinetics are consistent with structural studies which show that a conformational change occurs upon binding of methylthioacetate and other competitive inhibitors.
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Details
- Title
- Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase
- Creators
- Gouhua Zhao - Drexel UniversityMarilyn Schuman Jorns - Drexel University
- Publication Details
- Biochemistry (Easton), v 45(19), pp 5985-5992
- Publisher
- American Chemical Society; Washington, DC
- Grant note
- R01 GM031704 / NIGMS NIH HHS R01 GM031704-25 / NIGMS NIH HHS GM 31704 / NIGMS NIH HHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- [Retired Faculty]
- Web of Science ID
- WOS:000237579000007
- Scopus ID
- 2-s2.0-33646576258
- Other Identifier
- 991019168251304721
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- Web of Science research areas
- Biochemistry & Molecular Biology