| Abstract
| - Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzesthe oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8α-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as wellas the X-ray crystallographic characterization of three new enzyme·inhibitor complexes. MSOX stabilizesthe anionic form of the oxidized flavin (pKa = 8.3 versus 10.4 with free FAD), forms a thermodynamicallystable flavin radical, and stabilizes the anionic form of the radical (pKa< 6 versus pKa = 8.3 with freeFAD). MSOX forms a covalent flavin·sulfite complex, but there appears to be a significant kinetic barrieragainst complex formation. Active site binding determinants were probed in thermodynamic studies withvarious substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibitMSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed withsimple amines. The amino group of sarcosine is not essential, but binding affinity depends on the natureof the substitution (CH3XCH2CO2-, X = CH2< O < S < Se < Te), an effect which has been attributedto differences in the strength of donor−π interactions. MSOX probably binds the zwitterionic form ofsarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH3)2S+CH2CO2-]and dimethylglycine (Kd = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. Themethyl group of sarcosine is not essential but does contribute to binding affinity. The methyl groupcontribution varied from −3.79 to −0.65 kcal/mol with CH3XCH2CO2- depending on the nature of theheteroatom (NH2+> O > S) and appeared to be inversely correlated with heteroatom electron density.Charge-transfer complexes are formed with MSOX and CH3XCH2CO2- when X = S, Se, or Te. Anexcellent linear 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 identicallypositioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increasedatomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavieratoms. Although l-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (Kd, X=NH = 1.37 mM) andform charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O ≫ NH) islinearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.
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