Inactivation of monomeric sarcosine oxidase by reaction with N-(cyclopropyl)glycine.
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.
Zhao
G
G
Department of Biochemistry, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania 19129, USA.
Qu
J
J
Davis
F A
FA
Jorns
M S
MS
D000332:Aerobiosis; D000693:Anaerobiosis; D001407:Bacillus / Q000201:enzymology; D001894:Borohydrides / Q000737:chemistry; D003521:Cyclopropanes / Q000737:chemistry*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005415:Flavins / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011489:Protein Denaturation; D050879:Sarcosine Oxidase; D013053:Spectrophotometry
D001894:Borohydrides; D003521:Cyclopropanes; D004791:Enzyme Inhibitors; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; C025364:sodium borohydride; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D005998:Glycine