flavin-adenine-dinucleotide and sodium-borohydride

Monomeric sarcosine oxidase (MSOX) is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, such as l-proline. Our previous studies showed that N-(cyclopropyl)glycine (CPG) acts as a mechanism-based inactivator of MSOX [Zhao, G., et al. (2000) Biochemistry 39, 14341-14347]. The reaction results in the formation of a modified reduced flavin that can be further reduced and stabilized by treatment with sodium borohydride. The borohydride-reduced CPG-modified enzyme exhibits a mass increase of 63 +/- 2 Da as compared with native MSOX. The crystal structure of the modified enzyme, solved at 1.85 A resolution, shows that FAD is the only site of modification. The modified FAD contains a fused five-membered ring, linking the C(4a) and N(5) atoms of the flavin ring, with an additional oxygen atom bound to the carbon atom attached to N(5) and a tetrahedral carbon atom at flavin C(4) with a hydroxyl group attached to C(4). On the basis of the crystal structure of the borohydride-stabilized adduct, we conclude that the labile CPG-modified flavin is a 4a,5-dihydroflavin derivative with a substituent derived from the cleavage of the cyclopropyl ring in CPG. The results are consistent with CPG-mediated inactivation in a reaction initiated by single electron transfer from the amine function in CPG to FAD in MSOX, followed by collapse of the radical pair to yield a covalently modified 4a,5-dihydroflavin.

Topics: Borohydrides; Crystallography, X-Ray; Electron Transport; Flavin-Adenine Dinucleotide; Glycine; Molecular Structure; Oxidation-Reduction; Peptides, Cyclic; Sarcosine Oxidase

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.

Topics: Aerobiosis; Anaerobiosis; Bacillus; Borohydrides; Cyclopropanes; Enzyme Activation; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Flavins; Glycine; Kinetics; Models, Chemical; Oxidoreductases, N-Demethylating; Protein Denaturation; Sarcosine Oxidase; Spectrophotometry

Three different radioactively labeled N-(1-methylcyclopropyl)benzylamines [N-(1-Me)CBA] were synthesized and used to show which atoms of the inactivator remain bound to monoamine oxidase (MAO) after inactivation. Organic chemical reactions were employed to elucidate the structure of the enzyme adduct and clarify the mechanism of inactivation. Following inactivation and dialysis, the benzyl substituent is lost, but the methyl group and cyclopropyl carbons remain attached to the enzyme even after further dialysis against solutions containing 1 mM benzylamine or 8 M urea. Treatment of inactivated enzyme with sodium cyanoborohydride prior to dialysis results in the retention of the benzyl group, suggesting an imine linkage. One hydride from sodium boro[3H]hydride is incorporated into the dialyzed inactivated enzyme consistent with a ketone functional group. When Pronase-digested N-(1-Me)CBA-inactivated MAO is treated with basic potassium triiodide, iodoform is isolated, indicating the presence of a methyl ketone. During inactivation, the optical spectrum of the covalently bound active site flavin changes from that of oxidized to reduced flavin. After urea denaturation, the flavin remains reduced, suggesting covalent linkage of the inactivator to the cofactor. On the basis of previous results [Silverman, R. B., Hoffman, S. J., & Catus, W. B., III (1980) J. Am. Chem. Soc. 102, 7126-7128], it is proposed that the mechanism of inactivation involves transfer of one electron from N-(1-Me)CBA to the flavin, resulting in an amine radical cation and a flavin radical. Then, either the cyclopropyl ring is attacked by the flavin radical or the cyclopropyl ring opens, and the radical generated is captured by the flavin radical. The product of this mechanism is the imine of benzylamine and 4-flavinyl-2-butanone, the proposed enzyme-inactivator adduct.

Topics: Amines; Animals; Benzylamines; Binding Sites; Borohydrides; Cattle; Chemical Phenomena; Chemistry; Flavin-Adenine Dinucleotide; Mercaptoethanol; Mitochondria, Liver; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Swine