Wagner M A, Jorns M S
Department of Biochemistry, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania 19129, USA.
Biochemistry. 2000 Aug 1;39(30):8825-9. doi: 10.1021/bi000350y.
Monomeric sarcosine oxidase (MSOX) is a flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) to yield glycine, formaldehyde, and hydrogen peroxide. MSOX can oxidize other secondary amino acids (N-methyl-L-alanine, N-ethylglycine, and L-proline), but N,N-dimethylglycine, a tertiary amine, is not a substrate. N-Methyl-L-alanine is a good alternate substrate, exhibiting a k(cat) value (8700 min(-)(1)) similar to sarcosine (7030 min(-)(1)). Turnover with L-proline (k(cat) = 25 min(-)(1)) at 25 degrees C occurs at less than 1% of the rate observed with sarcosine. MSOX is converted to a two-electron reduced form upon anaerobic reduction with sarcosine or L-proline. No evidence for a spectrally detectable intermediate was obtained in reductive half-reaction studies with L-proline. The reductive half-reaction with L-proline at 4 degrees C exhibited saturation kinetics (k(lim) = 6.0 min(-)(1), K(d) = 260 mM) and other features consistent with a mechanism in which a practically irreversible reduction step (E(ox). S --> E(red).P) with a rate constant, k(lim), is preceded by a rapidly attained equilibrium (K(d)) between free E and the E.S complex. Steady-state kinetic studies with sarcosine and N-methyl-L-alanine in the absence or presence of a dead-end inhibitor (pyrrole-2-carboxylate) indicate that catalysis proceeds via a "modified" ping pong mechanism in which oxygen reacts with E(red).P prior to the dissociation of the imino acid product. In this mechanism, double reciprocal plots will appear nearly parallel (as observed) if the reduction step is nearly irreversible. A polar mechanism, involving formation of a covalent 4a-flavin-substrate adduct is one of several plausible mechanisms for sarcosine oxidation. Thiols are known to form similar 4a-flavin adducts. MSOX does not form a 4a-adduct with thioglycolate but does form a charge-transfer complex that undergoes an unanticipated one-electron-transfer reaction to yield the anionic flavin radical.
单体肌氨酸氧化酶（MSOX）是一种黄素酶，催化肌氨酸（N-甲基甘氨酸）的氧化脱甲基反应，生成甘氨酸、甲醛和过氧化氢。MSOX可以氧化其他仲氨基酸（N-甲基-L-丙氨酸、N-乙基甘氨酸和L-脯氨酸），但叔胺N,N-二甲基甘氨酸不是其底物。N-甲基-L-丙氨酸是一种良好的替代底物，其催化常数（k(cat)）值（8700 min⁻¹）与肌氨酸（7030 min⁻¹）相似。在25℃下，L-脯氨酸的周转速率（k(cat) = 25 min⁻¹）不到肌氨酸观察速率的1%。用肌氨酸或L-脯氨酸进行厌氧还原时，MSOX会转化为双电子还原形式。在用L-脯氨酸进行的还原半反应研究中，未获得光谱可检测中间体的证据。在4℃下用L-脯氨酸进行的还原半反应表现出饱和动力学（k(lim) = 6.0 min⁻¹，K(d) = 260 mM）以及其他与一种机制相符的特征，该机制中一个实际不可逆的还原步骤（E(ox).S → E(red).P），其速率常数为k(lim)，之前是游离E与E.S复合物之间快速达到的平衡（K(d)）。在有无终产物抑制剂（吡咯-2-羧酸盐）存在的情况下，用肌氨酸和N-甲基-L-丙氨酸进行的稳态动力学研究表明，催化作用通过一种“修正”的乒乓机制进行，其中氧气在亚氨基酸产物解离之前与E(red).P反应。在这种机制中，如果还原步骤几乎不可逆，双倒数图将几乎平行（如观察到的）。一种涉及形成共价4a-黄素-底物加合物的极性机制是肌氨酸氧化的几种可能机制之一。已知硫醇会形成类似的4a-黄素加合物。MSOX不会与巯基乙酸形成4a-加合物，但会形成一种电荷转移复合物，该复合物会发生意外的单电子转移反应，生成阴离子黄素自由基。
