Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants

Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme th at catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8 alpha-(S-cysteinyl)FAD]. This paper descr ibes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme inhibitor compl exes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical , and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Act ive site binding determinants were probed in thermodynamic studies with var ious substrate analogues whose binding was found to perturb the flavin abso rption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential fur binding since none is observed with simple amines. The ami no group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH3XCH2CO2-, X = CH2 < O < S < Se < Te), an ef fect which has been attributed to differences in the strength of donor-pi i nteractions. MSOX probably binds the zwitterionic form of sarcosine, as jud ged by the spectrally similar complexes formed with dimethylthioacetate [(C H3)(2)S+CH2CO2-] and dimethylglycine (K-d = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH3XCH2CO2- depending on the nature of the heteroatom (NH2+ > O > S) and appeared to be inversel y correlated with heteroatom electron density. Charge-transfer complexes ar e formed with MSOX and CH3XCH2CO2- when X = S, Se, or Te. An excellent line ar correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increas ed atomic radius of S < Se < Te appears to simultaneously favor an alternat e binding position for the heavier atoms. Although L-proline is a poor subs trate, aromatic heterocyclic carboxylates containing a five-membered ring a nd various heteroatoms (X = NH, O, S) are good ligands (K-d,K-X=NH = 1.37 m M) and form charge-transfer complexes with MSOX. The energy of the charge-t ransfer bands (S > O much greater than NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.