Identification of the Oxygen Activation Site in Monomeric Sarcosine Oxidase: Role of Lys265 in Catalysis

• Published in: Biochemistry (Easton) 2008-09, Vol.47 (35), p.9124-9135
• Main Authors: Zhao, Guohua, Bruckner, Robert C, Jorns, Marilyn Schuman
• Format: Article
• Language: English
• Subjects: Binding Sites; Catalysis; Kinetics; Lysine - chemistry; Lysine - genetics; Lysine - metabolism; Models, Molecular; Mutation; Oxidation-Reduction; Oxygen - chemistry; Oxygen - metabolism; Sarcosine - chemistry; Sarcosine - metabolism; Sarcosine Oxidase - chemistry; Sarcosine Oxidase - genetics; Sarcosine Oxidase - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi8008642

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 × 105 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.