Inactivation of Monomeric Sarcosine Oxidase by Reaction with N-(Cyclopropyl)glycine

Abstract

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound flavin adenine dinucleotide (FAD). The present study demonstrates that N-(cyclopropyl)glycine (CPG) is a mechanism-based inhibitor. CPG forms a charge transfer complex with MSOX that reacts under aerobic conditions to yield a covalently modified, reduced flavin (lambda(max) = 422 nm, epsilon(422) = 3.9 mM(-1) cm(-1)), accompanied by a loss of enzyme activity. The CPG-modified flavin is converted at an 8-fold slower rate to 1,5-dihydro-FAD (EFADH(2)), which reacts rapidly with oxygen to regenerate unmodified, oxidized enzyme. As a result, CPG-modified MSOX reaches a CPG-dependent steady-state concentration under aerobic conditions and reverts back to unmodified enzyme upon removal of excess reagent. No loss of activity is observed under anaerobic conditions where EFADH(2) is formed in a reaction that goes to completion at low CPG concentrations. Aerobic denaturation of CPG-modified enzyme yields unmodified, oxidized flavin at a rate similar to the anaerobic denaturation reaction, which yields 1,5-dihydro-FAD. The CPG-modified flavin can be reduced with borohydride, a reaction that blocks conversion to unmodified flavin upon removal of excess CPG or enzyme denaturation. The possible chemical mechanism of inactivation and structure of the CPG-modified flavin are discussed.

Oxidation of amines by flavoproteins

“…The monomeric sarcosine oxidase is slowly inactivated by N -cyclopropyl glycine [57]. In the process a modified flavin is formed that is unstable, but it can be reduced by sodium borohydride to a form sufficiently stable for structure determination [58].…”

Insights into the Mechanism of Flavoprotein-Catalyzed Amine Oxidation from Nitrogen Isotope Effects on the Reaction of N-Methyltryptophan Oxidase

“…Electron-transfer mechanisms have been frequently proposed for flavin-dependent amine oxidations (reviewed in (42)). Support for these mechanisms comes primarily from oxidation studies of cyclopropyl or cyclobutyl compounds that act as mechanism based inhibitors for MAO ((12) and references therein) and for MSOX (43,44). However, it is important to note that a cyclopropyl group blocks hydride transfer, as cyclopropylidene imines are very strained, and facilitates electron transfer, as cyclopropyl groups greatly stabilize adjacent positive charge.…”

pH and Kinetic Isotope Effects on Sarcosine Oxidation by N-Methyltryptophan Oxidase

“…Such a slow formation of an unstable neutral radical followed by a rapid electron transfer is effectively indistinguishable from a hydride transfer. which very slowly decomposes to fully reduced enzyme (21). While the structure of the modified cofactor was not determined, the inactivation was attributed to one-electron oxidation of the inhibitor to the cyclopropyl radical, which then reacted with the anionic flavin semiquinone.…”