Ionization of Zwitterionic Amine Substrates Bound to Monomeric Sarcosine Oxidase

Abstract

Monomeric sarcosine oxidase (MSOX) binds the L-proline zwitterion (pK a = 10.6). The reactive substrate anion is generated by ionization of the ES complex (pK a = 8.0). Tyr317 was mutated to Phe to determine whether this step might involve proton transfer to an active site base. The mutation does not eliminate the ionizable group in the ES complex (pK a = 8.9) but does cause a 20-fold decrease in the maximum rate of the reductive half-reaction. Kinetically determined K d values for the ES complex formed with L-proline agree with results obtained in spectral titrations with wild type or mutant enzyme. Unlike wild type enzyme, K d values with the mutant enzyme are pH-dependent, suggesting that the mutation has perturbed the pK a of a group that affects the K d . As compared with wild type enzyme, an increase in charge transfer band energy is observed for mutant enzyme complexes with substrate analogs while a 10-fold decrease in the charge transfer band extinction coefficient is found for the complex with L-proline anion. The results eliminate Tyr317 as a possible acceptor of the proton released upon substrate ionization. Since previous studies rule out the only other nearby base, we conclude that L-proline is the ionizable group in the ES complex and that amino acids are activated for oxidation upon binding to MSOX by stabilization of the reactive substrate anion. Tyr317 may play a role in substrate activation and optimizing binding, as judged by the effects of its mutation on the observed pK a , reaction rates and charge transfer bands.Monomeric sarcosine oxidase (MSOX) 1 is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] (1). The enzyme catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine) and the equivalent C-N bond in other secondary amino acids, such as L-proline. The crystal structure of free MSOX from Bacillus sp. B-0618 and complexes of the enzyme with various inhibitors have been determined (2-4). MSOX is a two-domain, 46 KDa protein with an overall topology similar to D-amino acid oxidase.MSOX is a member of a growing family of enzymes that contain covalently bound flavin and catalyze similar oxidation reactions with different amine substrates (5-8). Despite considerable attention, important questions regarding the mechanism of flavin-dependent amine oxidation reactions remain unresolved. Postulated mechanisms differ with respect to: 1) the requirement for an active site base; 2) the acceptor of the α-hydrogen removed from the carbon atom in the C-N bond undergoing oxidation; 3) geometric constraints on the orientation of flavin, substrate and a putative active site base (Scheme 1). In the hydride transfer mechanism, flavin N(5) acts as the acceptor of the α-hydrogen in a reaction that does not require an active site base. Single electron transfer (SET) mechanisms are initiated by electron transfer from substrate amino

Oxidation of amines by flavoproteins

“…Studies by Jorns and coworkers [52, 54, 55] of the effect of pH on the reductive half-reaction of the monomeric sarcosine oxidase are consistent with the enzyme being able to bind the substrate whether the nitrogen is neutral or charged, but further reaction only occurring from the anionic substrate. Thus, the anionic substrate is the active form, as is the case with DAAO.…”

Importance of glutamate 87 and the substrate α-amine for the reaction catalyzed by d-arginine dehydrogenase

“…Despite the presence of several groups in the active site of the enzyme with the potential to act as base, substrate deprotonation occurs through the direct release of the substrate a-amino proton to the solvent without involvement of protein residues. In this respect, PaDADH is similar to other well-characterized flavoproteins that catalyze the oxidation of amino acids, i.e., L-amino acid oxidase, DAAO, and monomeric sarcosine oxidase, which lack in their active sites catalytic bases [9,[30][31][32]. From a mechanistic standpoint, D-arginine, D-lysine and D-methionine, as well as their respective imino-products, are sticky, resulting in steady-state turnover being primarily populated of enzyme complexes with substrates and products rather than the free enzyme.…”

Spectral and Kinetic Characterization of the Michaelis Charge Transfer Complex in Monomeric Sarcosine Oxidase

“…We postulate that the ligand carboxylate in the initial MSOX complex "recruits" Arg52 in a process that results in the displacement of the two waters near Arg52 ( Figure 10, bottom panel, WAT 7,8) and conversion to a more stable closed complex. Kinetic and thermodynamic studies indicate that MSOX binds the zwitterionic form of L-proline (5,7). Substrate ionization may be promoted by hydrogen bond and electrostatic interactions at the active site that stabilize the anion.…”