CHROMATOGRAPHIC ANALYSIS OF THE CHIRAL AND COVALENT INSTABILITY OF S-ADENOSYL-L-METHIONINE
CHROMATOGRAPHIC ANALYSIS OF THE CHIRAL AND COVALENT INSTABILITY OF S-ADENOSYL-L-METHIONINE
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DOI：
10.1021/bi00363a041
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发表时间：
1986-07-29
期刊：
影响因子：
2.9
通讯作者：
HOFFMAN, JL
中科院分区：
文献类型：
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作者：
HOFFMAN, JL
具有生物活性的S-腺苷- l-蛋氨酸（AdoMet）的手性为S，S，其中的名称是指硫和。分别为碳。本文介绍了一种阳离子交换高效液相色谱（HPLC）分离（S，S）-AdoMet和（R，S）-AdoMet的方法。用该方法测定了（S，S）-AdoMet的降解速率随pH的变化规律，在37℃和pH 7.5条件下得到的一阶速率常数为外消旋1.8次。10-6s-1;裂解为同丝氨酸内酯和5“-（甲基硫）腺苷，4.6倍。10-6s-1;水解成腺嘌呤和s -戊基蛋氨酸3次。10-6s-1。在7.5 ~ 1.5的pH范围内，外消旋化速率没有变化。裂解反应一直持续到pH降至1.5，但水解在pH 6时停止。非放射性AdoMet的商业样品含有20-30%(R，S)-AdoMet，而[甲基- 3h]AdoMet的样品含有不到1% (R，S)-AdoMet。通过混合这些样品制备酶底物将导致比活性的低估和产物量的高估。小鼠肝脏的（R，S）-AdoMet/(S，S)-AdoMet比值为0.03，远小于上述速率常数计算的0.19。从小鼠肝脏提取的酶提取物不能降解（R，S）-ADoMet，但更彻底的研究可能会发现这种活性。无论如何，裂解和水解反应部分平衡了（S，S）-AdoMet在体内的外消旋，防止了（R，S）-AdoMet的过度积累。
The chirality of biologically active S-adenosyl-L-methionine (AdoMet) is S,S, where the designations refer to the sulfur and the .alpha.-carbon, respectively. This paper describes a cation-exchance high-performance liquid chromatographic (HPLC) method for separating (S,S)-AdoMet from the biologically inactive (R,S)-AdoMet that results from racemization at the sulfur. This method was used to measure the rates of the degradation reactions of (S,S)-AdoMet as a function of pH. these reactions and the first-order rate constants, which were found at 37.degree.C and pH 7.5, are racemization, 1.8 .times. 10-6s-1; cleavage to homoserine lactone and 5''-(methylthio)adenosine, 4.6 .times. 10-6s-1; and hydrolysis to adenine and S-pentosylmethionine, 3 .times. 10-6s-1. Racemization showed no change in rate over the pH range from 7.5 to 1.5. The cleavage reaction persisted until the pH was lowered to 1.5, but hydrolysis ceased at pH 6. Commercial samples of nonradioactive AdoMet contained 20-30%(R,S)-AdoMet, while a sample of [methyl-3H]AdoMet had less than 1% (R,S)-AdoMet. Preparing enzyme substrates by mixing such samples will cause an underestimate of specific activity and an overestimate of the amount of product. The (R,S)-AdoMet/(S,S)-AdoMet radio in mouse liver was 0.03, much less than the value of 0.19 calculated from the above rate constants. An enzyme extract from mouse liver did not degrade (R,S)-ADoMet, but a more thorough search may find such an activity. In any event, the cleavage and hydrolysis reactions partially balance the racemization of (S,S)-AdoMet in vivo and prevent excessive accumulation of (R,S)-AdoMet.