Ionization of zwitterionic amine substrates bound to monomeric sarcosine oxidase.

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 group to generate a flavin/substrate radical pair (9). Polar mechanisms involve reversible nucleophilic addition of substrate amino group at flavin C(4a) to yield a covalent flavin-substrate intermediate (10). An active site base is required for the versions of the SET and polar mechanisms shown in Scheme 1 but base-independent variants have been suggested where the α-hydrogen is transferred to flavin N(5) as a hydrogen atom (11) or proton (12), respectively. Scheme 1 Possible mechanisms for sarcosine oxidation by MSOX. IA is the imino acid product of sarcosine oxidation (CH2=NH+-CH2-CO2−); FADH− is the 1,5-dihydroflavin anion. With the possible exception of hydride transfer (13), it is generally agreed that the unprotonated amino group is the reactive species in flavin-dependent amine oxidation reactions. Since biologically relevant amines typically exhibit pKa values considerably above physiological pH, an active site base might be required to generate the unprotonated amine. Several observations indicate that MSOX binds the zwitterionic form of amino acid ligands. Although the enzyme is specific for secondary amines, tertiary amines like dimethylglycine act as competitive inhibitors. The crystallographic, spectral and theromodynamic properties of the MSOX·dimethylglycine complex strongly suggest that the inhibitor is bound as a zwitterion (4). The anaerobic half-reaction of MSOX with L-proline, a slow substrate, is rate-limiting during turnover and proceeds via a rapidly attained equilibrium (Kd) between free MSOX and the enzyme·substrate complex, followed by an essentially irreversible flavin reduction step (klim) (14) (Scheme 2). The kinetically determined Kd for the enzyme·L-proline complex is pH-independent in the pH range from 6.5 to 9.0. This indicates that MSOX binds the zwitterionic form of L-proline, the predominant species in solution at slightly acidic or alkaline pH values. Scheme 2 Proposed mechanism for the reductive half-reaction of MSOX with L-proline. In contrast, observed values for klim are strongly pH-dependent, indicating that an ionizable group in the enzyme·L-proline complex must be unprotonated prior to flavin reduction. The oxidized enzyme·L-proline complex exhibits an absorption band at longer wavelengths that is attributable to flavin-ligand charge transfer interaction. This feature is expected only for a complex of oxidized flavin with the electron-rich L-proline anion. Significantly, development of the charge transfer band is pH-dependent, exhibiting a spectrally detectable pKa (pKa = 7.9) that is virtually identical to the kinetically determined pKa for an ionizable group in the enzyme·substrate complex (pKa = 8.0) (14). The results suggest that the pKa of MSOX-bound L-proline may be decreased by more than 2.5 pH units as compared with free L-proline (pKa = 10.6). Alternatively, ionization of the protein-bound zwitterion may require proton transfer to an active site base with a pKa of 8.0. His269 or Tyr317 were identified as potential candidates for the active site base, as judged by the crystal structures observed for MSOX complexes with various inhibitors (2–4), including pyrrole-2-carboxylate (PCA) (Figure 1). In the MSOX·PCA complex, ND1 of His269 is located 4.17 A from the nitrogen in PCA and 3.6 A from the carbon atom in the inhibitor that corresponds to C(5) in L-proline or the methyl group in sarcosine. The corresponding distances for the phenolic oxygen of Tyr317 are 4.58 and 3.54 A, respectively. Mutation of His269 to Asn reduces the rate of MSOX turnover with sarcosine or the reductive half-reaction with L-proline by nearly 2 orders of magnitude but does not eliminate the ionizable group in the enzyme·substrate complex (3). In this paper, we characterize the effect of mutating Tyr317 to Phe on MSOX catalysis. Figure 1 Stereoview of the active site in the MSOX complex with pyrrole-2-carboxylate (PCA). The figure was constructed using the previously reported structure (2). Atoms are colored according to the following colors: carbon, green; oxygen, red; nitrogen, blue. ...