Akhtar M, Ajaz A A, Corina D L
Biochem J. 1984 Nov 15;224(1):187-94. doi: 10.1042/bj2240187.
The mechanism through which the C-17(3) carboxy group of bacteriochlorophyllide a is esterified to produce bacteriochlorophyll aphytyl of Rhodopseudomonas spheroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum was studied by using 5-aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms. The latter species was prepared by an exchange reaction in which 5-aminolaevulinate hydrochloride was heated in H218O in an autoclave. A method for the determination of the 18O content of the C-1 oxygen atoms of 5-aminolaevulinate was developed. As a prelude to the mechanistic work, a systematic study was undertaken to establish the optimal conditions under which a significant proportion of the bacteriochlorophyll a of the two photosynthetic organisms originated from the exogenously added 5-aminolaevulinate. It was found that, when Rps. spheroides and Rsp. rubrum were grown in the presence of about 0.15mM- and 1.2mM-5-aminolaevulinate respectively, 30-40% of their chlorophyll was derived from the added precursor. In these conditions, 5-amino[1,4-18O3]laevulinate was incorporated into bacteriochlorophyll aphytyl and bacteriochlorophyll ageranylgeranyl by the relevant organisms. The samples of chlorophylls were then hydrolysed with alkali to obtain phytol and geranylgeraniol, which were converted into the corresponding trimethylsilyl derivatives and analysed by gas chromatography-mass spectrometry. The data were used to deduce that the alcohols contained 90-95% of the 18O originally present at each of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results it is suggested that the ester bond at C-17(3) is formed, not by a chlorophyllase type of enzymic reaction, but by a process involving the nucleophilic attack by the C-17(3) carboxylate group of the chlorophyllide on the activated form of an isoprenyl alcohol.
利用在其C-1羧基氧原子处用¹⁸O标记的5-氨基乙酰丙酸，研究了细菌叶绿素a的C-17(3)羧基被酯化以生成球形红假单胞菌的细菌叶绿素植基酯和深红红螺菌的细菌叶绿素香叶基香叶酯的机制。后一种物质是通过交换反应制备的，其中盐酸5-氨基乙酰丙酸在高压釜中于H₂¹⁸O中加热。开发了一种测定5-氨基乙酰丙酸C-1氧原子¹⁸O含量的方法。作为机理研究的前奏，进行了一项系统研究，以确定最佳条件，在该条件下，两种光合生物的大部分细菌叶绿素a源自外源添加的5-氨基乙酰丙酸。结果发现，当球形红假单胞菌和深红红螺菌分别在约0.15mM和1.2mM的5-氨基乙酰丙酸存在下生长时，它们30-40%的叶绿素源自添加的前体。在这些条件下，5-氨基[1,4-¹⁸O₃]乙酰丙酸被相关生物体掺入细菌叶绿素植基酯和细菌叶绿素香叶基香叶酯中。然后用碱水解叶绿素样品以获得叶绿醇和香叶基香叶醇，将它们转化为相应的三甲基硅烷基衍生物并通过气相色谱-质谱法进行分析。这些数据用于推断这些醇含有最初存在于前体5-氨基乙酰丙酸每个C-1氧原子处90-95%的¹⁸O。根据这些结果表明，C-17(3)处的酯键不是通过叶绿素酶类型的酶促反应形成的，而是通过叶绿素酸的C-17(3)羧酸盐基团对异戊二烯醇活化形式的亲核攻击过程形成的。
