Discovery of a third coenzyme in sarcosine oxidase.
Discovery of a third coenzyme in sarcosine oxidase.
复制标题
肌氨酸氧化酶中第三种辅酶的发现。
DOI：
10.1021/bi00051a019
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Jorns,MS
中科院分区：
文献类型：
--
作者：
Willie,A;Jorns,MS
重组肌氨酸氧化酶或从棒状杆菌P-1分离的天然酶用盐酸胍变性释放非共价结合的FAD和第二个UV吸收组分（峰2），其在反相HPLC期间与NAD+共迁移。FAD和峰2也在通过在37 ℃下孵育肌氨酸氧化酶30分钟制备的提取物中发现，该过程导致酶的非共价结合的FAD的部分（~ 50%）释放。37 ℃提取物中的峰2是热不稳定的，在pH 8.0下煮沸5分钟后分解。用NAD+观察到类似的不稳定性。来自肌氨酸氧化酶的37 ℃提取物与磷酸二酯酶反应产生烟酰胺单核苷酸肽、AMP和FMN，如对于含有NAD+和FAD的混合物所预期的。当37 ℃提取物与酵母醇脱氢酶在乙醇存在下反应时，峰2转化为NADH。由重组或天然酶制备的盐酸胍提取物含有1 mol NAD+/mol FAD。由于肌氨酸氧化酶含有1摩尔非共价结合的FAD，结果表明该酶还含有1摩尔NAD+。NAD+紧密结合，在酶纯化过程中不会丢失。它不易被NAD酶水解、被醇脱氢酶还原或被氰化物亲核攻击。与肌氨酸氧化酶中的黄素不同，NAD+不被肌氨酸还原，也不与黄素处于氧化还原平衡状态。当棒状杆菌P-1以肌氨酸作为碳源和能量源生长时，肌氨酸氧化酶作为诱导酶产生。在O2和NH 4-叶酸存在下，1该酶催化肌氨酸的氧化去甲基化以产生甘氨酸、过氧化氢和5，10-CH 2-NH 4叶酸（当量1）。（甲醛生成到位
Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Coryne-bacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 C for 30 min, a procedure which causes partial (~ 50%) release of the enzyme’s noncovalently bound FAD. Peak 2 in the 37 C extract is heat labile and decomposes upon boiling for 5 min at pH 8.0. A similar instability was observed with NAD+. Reaction of the 37 C extract from sarcosine oxidase with phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, prepared from recombinant or natural enzyme, contain 1 mol of NAD+/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD+ is not reduced by sarcosine and is not in redox equilibrium with the flavins.Sarcosine oxidase is produced as an inducible enzyme when Corynebacterium sp. P-1 is grown with sarcosine as source of carbon and energy. In the presence of 02 and H4-foíate, 1 the enzyme catalyzes the oxidative demethylation of sarcosine to yield glycine, hydrogen peroxide, and 5, 10-CH2-H4folate (eq 1).(Formaldehyde is generated in place