title: Monomeric sarcosine oxidase: evidence for an ionizable group in the E.S complex.
abstract: Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, including L-proline. The reductive half-reaction with L-proline proceeds via a rapidly attained equilibrium (K(d)) between free E(ox) and the E(ox).S complex, followed by a practically irreversible reduction step (E(ox).S --> E(red).P) associated with a rate constant, k(lim). The effect of pH on the reductive half-reaction shows that the K(d) for L-proline binding is pH-independent (pH 6.46-9.0). This indicates that MSOX binds the zwitterionic form of L-proline, the predominant species in solution at neutral pH (pK(a) = 10.6). Values for the limiting rate of reduction (k(lim)) are, however, strongly pH-dependent and indicate that an ionizable group in the E(ox).L-proline complex (pK(a) = 8.02) must be unprotonated for conversion to E(red).P. Charge-transfer interaction with L-proline as the donor and FAD as acceptor is possible only with the anionic form of L-proline. The ionizable group in the E(ox).L-proline complex is required for conversion of enzyme-bound L-proline from the zwitterionic to the reactive anionic form, as judged by the independently determined pK(a) for charge-transfer complex formation with the MSOX flavin (pK(a) = 7.94). The observation that the anionic form of L-proline with a neutral amino group is the reactive species in the reduction of MSOX is similar to that observed for other flavoenzymes that oxidize amines, including monoamine oxidase and trimethylamine dehydrogenase.
authors.affiliation: Department of Biochemistry, MCP Hahnemann School of Medicine, Philadelphia, PA 19129, USA.
authors.forename: Gouhua
authors.initials: G
authors.lastname: Zhao
authors.forename: Marilyn Schuman
authors.initials: MS
authors.lastname: Jorns
mesh_terms: D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry*; D050879:Sarcosine Oxidase; D013057:Spectrum Analysis
chemical_list: D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase