Journal article
Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis
Biochemistry (Easton), v 47(35), pp 9124-9135
02 Sep 2008
PMID: 18693755
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme.sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 x 10(5) M(-1) s(-1)) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.
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Details
- Title
- Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis
- Creators
- Guohua Zhao - Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USARobert C BrucknerMarilyn Schuman Jorns
- Publication Details
- Biochemistry (Easton), v 47(35), pp 9124-9135
- 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
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:000258721200010
- Scopus ID
- 2-s2.0-50849101652
- Other Identifier
- 991014878092904721
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- Web of Science research areas
- Biochemistry & Molecular Biology