乙酰乙酰辅酶a结构式
乙酰辅酶a三羧酸循环产物_概述及解释说明
乙酰辅酶a三羧酸循环产物概述及解释说明1. 引言1.1 概述乙酰辅酶A三羧酸循环是生物体内一种重要的代谢途径，也被称为柠檬酸循环或Krebs循环。
它是将食物中的营养成分（如葡萄糖、脂肪和氨基酸）转化为能量的关键过程之一。
该循环产生多种有机化合物作为产物，这些产物在维持机体正常功能和能量供应方面起着至关重要的作用。
1.2 文章结构本文将首先对乙酰辅酶A三羧酸循环产物进行定义和背景介绍，然后详细阐述其关键反应与中间产物。
接着，我们将探讨这些产物在生理上的意义与作用，并解释其形成过程。
此外，文章还将涵盖能量生成与转移途径以及与乙酰辅酶A 三羧酸循环相关的调控机制和相关疾病。
最后，我们总结了乙酰辅酶A三羧酸循环产物的重要性，并展望了未来的研究方向和应用前景。
1.3 目的本文的目的是为读者提供一个全面而系统的对乙酰辅酶A三羧酸循环产物的概述和解释说明。
通过深入了解这些产物在代谢过程中所扮演的角色，我们可以更好地理解生物体内能量转化和调控机制，并为相关疾病的治疗和未来研究提供新的思路和方向。
2. 乙酰辅酶a三羧酸循环产物2.1 定义和背景乙酰辅酶A三羧酸循环（TCA循环），也称为柠檬酸循环或Krebs循环，是细胞代谢中的一个重要过程。
TCA循环发生在细胞的线粒体内，并通过一系列反应将营养物质转化为能量。
TCA循环的产物是一系列有机化合物，其中包括柠檬酸、丙二酸、异柠檬酸、脱氧核糖核苷二磷酸（NADH）、转移磷酮选择半透膜肾上腺素受体（FADH2）、三羟基丁二醛等。
2.2 关键反应与中间产物TCA循环是一个复杂的过程，包括多个关键反应和中间产物。
首先，在糖类、脂肪和氨基酸代谢过程中生成的乙酰辅酶A进入TCA循环并与草酰乙二磷脂结合形成柠檬酸。
随后，柠檬酸通过一系列反应逐步转化为丙二酸、异柠檬酸和脱氧核糖核苷二磷酸（NADH）等产物。
最终，乙酰辅酶A再次生成并参与下一个循环。
2.3 生理意义与作用TCA循环产物在细胞代谢中起着重要的作用。
乙酰辅酶a彻底氧化的磷氧比
乙酰辅酶A（Acetyl-CoA）是生物体内能量代谢的重要中间产物，其彻底氧化的过程涉及到多个步骤，包括脂肪酸的β-氧化、酮体的生成和氧化、以及三羧酸循环等。
在这个过程中，磷氧比（P/O比）是一个关键的参数，它反映了细胞内能量代谢的效率。
乙酰辅酶A的彻底氧化过程可以分为两个阶段：第一阶段是脂肪酸的β-氧化，这个阶段在细胞的线粒体中进行，每氧化一个碳原子需要消耗四个氢原子和一个电子，同时产生三个NADH和一个FADH2。
第二阶段是三羧酸循环，这个阶段在线粒体的基质中进行，每氧化一个碳原子需要消耗两个氢原子和一个电子，同时产生一个NADH和一个FADH2。
在这两个阶段中，磷氧比的变化主要取决于NADH和FADH2的数量。
NADH和FADH2是细胞内的主要电子供体，它们在电子传递链中通过一系列的氧化还原反应将电子传递给氧气，从而产生ATP。
在这个过程中，每传递四个电子，就会消耗一个氧气分子，产生一个水分子。
因此，NADH和FADH2的数量越多，产生的ATP就越多，磷氧比也就越高。
然而，NADH和FADH2的数量并不是随意的，它们受到许多因素的影响，包括乙酰辅酶A 的来源、脂肪酸的种类和长度、三羧酸循环的速度等。
例如，如果乙酰辅酶A主要来自于脂肪酸的β-氧化，那么产生的NADH和FADH2就会比较多，磷氧比也就比较高；反之，如果乙酰辅酶A主要来自于酮体的生成和氧化，那么产生的NADH和FADH2就会比较少，磷氧比也就比较低。
此外，脂肪酸的种类和长度也会影响磷氧比。
一般来说，长链脂肪酸的β-氧化速度比较慢，产生的NADH和FADH2也就比较少；而短链脂肪酸的β-氧化速度比较快，产生的NADH和FADH2也就比较多。
因此，长链脂肪酸的磷氧比通常会比较低。
总的来说，乙酰辅酶A的彻底氧化过程中的磷氧比是一个复杂的参数，它受到许多因素的影响。
通过调控这些因素，可以提高细胞内能量代谢的效率，从而提高生物体的生存和繁殖能力。
乙酰辅酶a转化为酮体的原因
乙酰辅酶a转化为酮体的原因
乙酰辅酶A（Acetyl-CoA）是一种重要的代谢中间产物，它可
以通过多种途径转化为酮体。
以下是乙酰辅酶A转化为酮体的原因：
1. 饥饿状态，在饥饿状态下，人体的血糖水平下降，胰岛素分
泌减少，导致葡萄糖供应不足。
这时，肝脏开始分解脂肪酸产生乙
酰辅酶A，进而通过肝脏中的酮体生成途径（Ketogenesis）转化为
酮体（如β-羟基丁酸、乙酰乙酸等），以提供额外的能量供给身
体其他组织。
2. 高脂饮食，摄入高脂饮食会导致体内乙酰辅酶A的增加，因
为脂肪酸经过β氧化分解后会生成乙酰辅酶A，再进一步转化为酮体。
这也是为什么一些严格的低碳水化合物高脂饮食（如减肥饮食）会诱导体内酮体生成的原因。
3. 糖尿病酮症酸中毒，在糖尿病患者中，由于胰岛素分泌不足
或者胰岛素抵抗，细胞无法充分利用葡萄糖，导致血糖升高，同时
也促进了脂肪酸的分解，使乙酰辅酶A增加，进而增加酮体的生成。
如果酮体生成过多，会导致糖尿病酮症酸中毒，这是一种严重的并
发症。
总的来说，乙酰辅酶A转化为酮体的原因主要与饥饿状态、高脂饮食以及糖尿病等代谢状态有关。
这些情况下，乙酰辅酶A的积累促进了酮体的生成，从而满足身体对能量的需求或代谢状态的调节。
希望这些信息能够回答你的问题。
邢其毅《基础有机化学》笔记和课后习题(含考研真题)详解(萜类化合物、甾族化合物和生物碱)
第23章萜类化合物、甾族化合物和生物碱23.1 复习笔记一、萜类化合物1．萜类化合物的生物合成萜类（terpene）化合物是广泛分布于植物、昆虫、微生物等动植物体内的一类有机化合物。
在生物体内，萜类化合物是由乙酰辅酶A（简写为CH3COSCoA）转化而来的。
乙酰辅酶A的结构简式如下所示：（1）转化过程：首先乙酰辅酶A和二氧化碳结合转化为丙二酰辅酶A，后者再和一分子的乙酰辅酶A形成乙酰乙酰辅酶A，这个中间体再和一分子乙酰辅酶A进行羟醛缩合反应，就得一个六碳中间体，然后还原水解，产生萜的生物合成前体，3-甲基-3，5-二羟基戊酸。
乙酰辅酶A 丙二酰辅酶A 乙酰乙酰辅酶A六碳中间体3-甲基-3，5-二羟基戊酸（2）3-甲基-3，5-二羟基戊酸是生物合成的一个有效前体。
（3）由3-甲基-3，5-二羟基戊酸变为异戊二烯体系还需要失去一个碳原子。
经过腺苷三磷酸酯（ATP）的作用，两个羟基分步骤地进行磷酸化，然后失去磷酸，同时失羧，得到焦磷酸异戊烯酯（isopentenyl pyrophosphate）。
（4）由焦磷酸异戊烯酯再进行结合就可生成各种萜类化合物。
例如拢牛儿醇（geraniol）的生成过程如下所示：2．萜类化合物的结构组成和分类（1）结构组成①这些分子可以看作是两个或两个以上的异戊二烯分子，以头尾相连的方式结合起来的。
例如在对烷分子中，是由一个异戊二烯分子中的C-1和另一个异戊二烯分子的C-4′结合起来的：②异戊二烯规则（isoprene rule）。
现在已知：绝大多数萜类分子中的碳原子数目是异戊二烯五个碳原子的倍数，仅发现个别的例外。
（2）萜类化合物可以根据组成分子中异戊二烯单位的数目来分类。
碳原子数异戊二烯单位单萜l0 2倍半萜15 3二萜20 4三萜30 6多萜3．萜类化合物的实例（1）单萜从植物的花、叶、果皮、种子及树皮等中提取得到的具有芳香气味的易挥发的液体称为精油。
单萜类化合物是精油的主要成分之一。
乙酰辅酶a彻底氧化的磷氧比
乙酰辅酶a彻底氧化的磷氧比乙酰辅酶A（Acetyl-CoA）是一个在细胞代谢中起关键作用的辅酶。
它可以通过氧化而彻底分解为二氧化碳和水，并释放出大量的能量。
乙酰辅酶A的彻底氧化过程被称为三羧酸循环（TCA循环），也被称为克雷布斯循环。
TCA循环是一种在细胞线粒体中进行的代谢途径，它包含了一系列复杂的化学反应。
整个过程需要多个酶的参与，并且分为一系列的步骤进行。
乙酰辅酶A进入TCA循环后，经过一系列的氧化和还原反应，最终完全氧化为二氧化碳和水。
首先，乙酰辅酶A在TCA循环开始时与草酰乙酸（oxaloacetate）结合，形成柠檬酸（citrate）。
柠檬酸随后经历一系列酸化和脱羧反应，生成丙酮酸（pyruvate）。
接着，丙酮酸进一步被氧化为乳酸（lactate），释放出能量。
接下来，乳酸被进一步氧化为丙酮酸。
丙酮酸进入TCA循环后，通过一系列反应逐渐被氧化。
在这个过程中，乙酰辅酶A释放出电子，并通过酶的参与，被导入到线粒体内的电子传递链中。
电子传递链是一个位于线粒体内膜上的复合物系统，它能够利用乙酰辅酶A释放的电子产生化学能。
在电子传递链中，电子从一个底物移动到另一个底物，产生了一系列的还原和氧化过程。
这些过程释放出的能量被用来驱动质子泵向线粒体内膜的外侧泵出氢离子（H+），形成质子梯度。
质子梯度可以用来产生ATP分子，ATP是细胞内储存和传递能量的主要分子。
通过线粒体内膜上的酶，质子可以通过ATP合酶被导回到线粒体内膜的内侧，从而产生ATP。
这个过程被称为氧化磷酸化，是乙酰辅酶A彻底氧化的过程中的一个重要环节。
当乙酰辅酶A被氧化完成后，最终生成的产物为二氧化碳和水。
二氧化碳可以通过呼吸作用被排出体外，而水则可以被细胞利用。
彻底氧化乙酰辅酶A所释放的能量也用于维持细胞的生命活动和进行其他代谢过程。
总的来说，乙酰辅酶A的彻底氧化过程是一个复杂的过程，需要多个酶的协同作用。
通过氧化乙酰辅酶A，细胞可以从中释放出大量的化学能，并将其转化为ATP储存起来，以供细胞对各种生物学过程的需要。
乙酰辅酶A
Volatile Ester Formation in Roses.Identification of an Acetyl-Coenzyme A.Geraniol/Citronellol Acetyltransferase in Developing Rose Petals1Moshe Shalit,Inna Guterman,Hanne Volpin,Einat Bar,Tal Tamari,Naama Menda,Zach Adam,Dani Zamir,Alexander Vainstein,David Weiss,Eran Pichersky,and Efraim Lewinsohn*Department of Vegetable Crops,Newe Ya’ar Research Center,Agricultural Research Organization,P.O.Box 1021,Ramat Yishay30095,Israel(M.S.,E.B.,T.T.,E.L.);The Institute of Plant Sciences and Genetics in Agriculture,Faculty of Agricultural,Food,and Environmental Quality Sciences,The Hebrew University of Jerusalem,P.O.Box12,Rehovot76100,Israel(M.S.,I.G.,N.M.,Z.A.,D.Z.,A.V.,D.W.);Department of Molecular,Cellular,and Developmental Biology,University of Michigan,830North University Street,Ann Arbor,Michigan48109–1048(E.P.);and Bioinformatics,Department of Genomics,Agricultural Research Organization,Volcani Center,Bet Dagan50250,Israel(H.V.)The aroma of roses(Rosa hybrida)is due to more than400volatile compounds including terpenes,esters,and phenolic derivatives.2-Phenylethyl acetate,cis-3-hexenyl acetate,geranyl acetate,and citronellyl acetate were identified as the main volatile esters emitted by the flowers of the scented rose var.“Fragrant Cloud.”Cell-free extracts of petals acetylated several alcohols,utilizing acetyl-coenzyme A,to produce the corresponding acetate esters.Screening for genes similar to known plant alcohol acetyltransferases in a rose expressed sequence tag database yielded a cDNA(RhAAT1)encoding a protein with high similarity to several members of the BAHD family of acyltransferases.This cDNA was functionally expressed in Escherichia coli,and its gene product displayed acetyl-coenzyme A:geraniol acetyltransferase enzymatic activity in vitro.The RhAAT1protein accepted other alcohols such as citronellol and1-octanol as substrates,but2-phenylethyl alcohol and cis-3-hexen-1-ol were poor substrates,suggesting that additional acetyltransferases are present in rose petals.The RhAAT1 protein is a polypeptide of458amino acids,with a calculated molecular mass of51.8kD,pI of5.45,and is active as a monomer.The RhAAT1gene was expressed exclusively in floral tissue with maximum transcript levels occurring at stage 4of flower development,where scent emission is at its peak.Roses(Rosa hybrida)are grown as garden plants,for the cut-flower industry,and as a source of natural fragrances.Many modern cut-flower rose cultivars were selected for long vase life,flower shape,and color.Intensive breeding has also generated garden cultivars that have an intense“rose”scent.More than 400different volatile compounds have been identi-fied in rose scent,and these compounds have been classified into several chemical groups including hy-drocarbons,alcohols,esters,aromatic ethers,and “others”(aldehydes such as geranial and nonanal, rose oxide,and norisoprenes such as-ionone;Fla-ment et al.,1993;Weiss,1997).Volatile esters such as geranyl acetate and 2-phenylethyl acetate are important contributors to the aroma of roses and many other flowers(Knudsen and Tollsten,1993).Volatile esters also contribute to the unique aroma of fruits such as banana(Musa acuminata),apple(Malus domestica),melon(Cucumis melo),strawberry(Fragaria ananassa),and spice plants such as lavender(Lavandula officinalis;Croteau and Karp,1991;Ueda et al.,1992).Despite the knowledge of the contribution of volatile acetate esters to the aroma of roses,including the demonstration of the circadian emission of some major volatile acetate es-ters by flowers of the rose var.“Honesty”(Helsper et al.,1998),little is known about the mechanisms by which these compounds are formed in roses. Acetate esters in plants are normally generated as a result of the action of alcohol acetyltransferase(AAT) enzymes that transfer the acetyl moiety from acetyl-CoA to an alcoholic substrate(Fig.1;Harada et al., 1985;Fellman and Mattheis,1995;Perez et al.,1996; Aharoni et al.,2000;Shalit et al.,2001).To date,only two genes that code for AAT enzymes involved in volatile esters formation in flowers have been iden-tified(Dudareva et al.,1998;D’Auria et al.,2002). They include the gene BEAT,which codes for a ben-zyl AAT,and the gene BEBT,which codes for a benzyl alcohol benzoyl transferase.The gene prod-ucts generate benzyl acetate and benzylbenzoate,re-spectively,two important volatiles emitted from the1This work was supported by the Israeli Ministry of Sciences, Culture,and Sport(grant no.1410–2–00to E.L.,D.Z.,Z.A.,A.V., and D.W.),and by a BARD scholarship(to E.P.).This is publication no.143/2002of the Agricultural Research Organization(Bet Da-gan,Israel).*Corresponding author;e-mail twefraim@.il; fax972–4–983–6936.Article,publication date,and citation information can be found at /cgi/doi/10.1104/pp.102.018572.flowers of Clarkia breweri .BEAT and BEBT both be-long to the BAHD family of acyltransferases,a group of monomeric enzymes of roughly 450amino acids whose two main hallmarks are the HXXXD motif believed to constitute the active site and an addi-tional motif,DFGWG,of unknown function (St-Pierre and De Luca,2000).The floral scent of “Fragrant Cloud,”a rose garden variety that is profoundly scented,consists largely of esters (mainly acetate derivatives),aromatic alcohols (mainly 2-phenylethyl alcohol),monoterpene alco-hols (such as citronellol and geraniol),and other monoterpenes and sesquiterpenes (primarily germa-crene D;Flament et al.,1993;Guterman et al.,2002).To study volatile ester formation in roses,we have followed an integrated approach,combining data ob-tained from the analysis of volatile composition in the headspace of flowers,together with enzymatic activity data from petal cell-free extracts,bioinfor-matic analysis of expressed sequence tag (EST)data-bases,and functional expression of candidate genes in Escherichia coli .This approach has already led to the isolation and characterization of several genes involved in biosynthesis of terpenoid and methyl-ether volatile components of the rose floral scent (Channeliere et al.,2002;Guterman et al.,2002;Lavid et al.,2002).Here,we describe the determination of the main volatile acetate esters present in “Fragrant Cloud ”roses headspace,the identification of AAT activities involved in volatile acetate formation,and the isolation and characterization of a new gene,highly expressed in developing rose petals,which encodes a protein that catalyzes the formation gera-nyl acetate in vitro.RESULTSVolatile Acetate Esters Are Major Constituents of “Fragrant Cloud”Floral ScentThe levels of volatile acetate esters emitted from flower buds (stages 1and 2)of “Fragrant Cloud ”roses are very low (0.4g per flower per 24h and 0.2g per flower per 24h,respectively)and consist almost exclusively of cis -3-hexenyl acetate (Fig.2A).During flower opening,the level of emission of ace-tate esters increased to 50g per flower per 24h at stage 3and 90g per flower per 24h at stage 4,reaching maximal levels of 200g per flower per 24h at fully open flowers (stage 5)and decreasing to only 15g per flower per 24h in fully open flowers (stage 6;Fig.2A).2-Phenylethyl acetate and cis -3-hexenyl acetate were the major acetates emitted.Geranyl ace-tate,citronellyl acetate,hexyl acetate,and 2-hexenyl-acetate were also abundant,whereas benzyl acetate and neryl acetate were emitted only at low levels.Although the total levels of emission of acetate esters changed during development,the ratios between the different acetate esters in each flowering stage (except when the flowers are still closed,stages 1and 2)were generally constant throughout flower opening.At stage 6,only 2-phenylethyl,cis -3-hexenyl,and hexyl acetate werenoted.Figure 1.Formation of volatile esters by AAT enzymatic activity.AAT enzyme catalyzes the formation of esters using acetyl-CoA and alcohols as the substrates.A few of the main substrates of these enzymes areshown.Figure 2.Emission of acetate esters from intact flowers (A)and alcohol:acetyl-CoA enzymatic activity levels in cell-free extracts of rose petals (B and C).Emission of acetate esters from “Fragrant Cloud”flowers was assessed by the headspace technique (A).Means of five to 10determinations per flower stage are given.The levels of AAT activity in cell-free extracts utilizing the monoterpenoids ge-raniol,citronellol or nerol as substrates (B),and the aromatic 2-phenylethyl alcohol or the aliphatic cis -3-hexenyl-1-alcohol as substrates (C).Means and SE of two replicates,each representing an individual flower.The experiment was replicated three times with similar results.Geraniol/Citronellol Acetyltransferase in Rose PetalsAAT Activities in Cell-Free ExtractsDerived from PetalsCell-free extracts from open flowers were prepared to evaluate the possible involvement of AATs in the formation of volatile acetate esters.Alcoholic sub-strates putatively involved in the formation of the major acetate esters emitted in“Fragrant Cloud”open flowers(stages4and5)were tested(Table I). The highest levels of AAT enzymatic activity were detected with medium-chain aliphatic alcohols such as cis-3-hexene1-alcohol and1-hexanol(237%and 204%,respectively,as compared with the rates ob-tained with geraniol).High activity(80%–150%as compared with the rate obtained for geraniol)was also detected when medium-chain aliphatic1-octanol and the aromatic alcohol2-phenylethyl alcohol,and the aliphatic isoamyl alcohol and butanol and the monoterpene citronellol were used as substrates. Moderate levels of activity(30%–50%of the rate of geraniol)were observed when the monoterpene al-cohol nerol,the medium-chain aliphatic1-decanol, and the aromatic benzyl alcohol were used as sub-strates.Relatively low levels of activity(15%–20%of the rate of geraniol)were detected with ethanol,and with linalool,a tertiary monoterpene alcohol.To test for changes in AAT enzymatic activities in rose petals during flower development,cell-free ex-tracts of petals of the six flowering stages were pre-pared and tested for potential enzymatic acetylation activity with five putative alcohol substrates(Fig.2,B and C).Enzymatic activity leading to the synthesis of geranyl acetate and citronellyl acetate from the monoterpene alcohols geraniol and citronellol,re-spectively,moderately increased during flower devel-opment,reaching a maximum level of about15pkat mgϪ1protein for geranyl acetate formation at stages3 and4and a maximum of12pkat mgϪ1protein at stage 4for citronellyl acetate formation(Fig.2B).In contrast, the potential for neryl acetate formation was low dur-ing all flower developmental stages.In addition,AAT activities with2-phenylethyl alcohol and cis-3-hexene 1-alcohol,which led to the formation of2-phenylethyl acetate and cis-3-hexenyl acetate,respectively,were already high at stage1(20pkat mgϪ1protein and55 pkat mgϪ1protein,respectively;Fig.2C),reached maximal levels at stages2to3(35pkat mgϪ1protein for2-phenylethyl alcohol and60pkat mgϪ1protein for cis-3-hexene1-alcohol),and subsequently declined at stages4to6to lower levels than those seen in the first stages(15pkat mgϪ1protein for2-phenylethyl alcohol and25pkat mgϪ1protein for cis-3-hexene1-alcohol).Isolation and Characterization of RhAAT1,an AAT Gene,from“Fragrant Cloud”PetalsAn EST database of“Fragrant Cloud”petals that contains more than1,834unigenes has been estab-lished(Guterman et al.,2002).A search in this data-base for genes encoding potential AAT enzymes high-lighted three ESTs displaying sequence similarity with known BAHD AAT genes from other sources.One such cDNA,which we designated as RhAAT1,en-codes a polypeptide of458amino acid residues(Fig.3) with a calculated molecular mass of51.8kD and a pI of5.45.The protein contains the two highly conserved motifs HXXXD and DFGWG present in the enzymes of the plant BAHD O-acyltransferases family.The protein sequence shows the highest identity, 69%,to a strawberry protein designated SAAT, which was isolated from ripening strawberry fruits and shown to catalyze the formation of medium-chain aliphatic acetate esters(Aharoni et al.,2000). RhAAT1is also31%identical to the protein encoded by the benzyl AAT gene(BEAT),which catalyzes the formation of benzyl acetate in flowers of C.breweri (Dudareva et al.,1998).RhAAT1was highly similar(E valueϭ10Ϫ49)to the pfam transferase(PF02458)domain and is also rela-tively closely related to two other enzymes of the BAHD plant acetyltransferases.It is31%identical to DAT,an acetyltransferase involved in vindoline for-mation in Catharanthus roseus(St-Pierre et al.,1998; Laflamme et al.,2001)and27%identical to TAT,an acetyltransferase involved in taxol formation in Taxus cuspidata(Walker et al.,2000;Fig.3B).Substrate Preference of the RecombinantRhAAT1EnzymeThe coding region of RhAAT1was subcloned into a pET(11a)expression vector(Studier et al.,1990; Lavid et al.,2002)for functional expression in E.coli.Table I.Relative alchohol acetyltransferase activity of crudeϩ3ϩ95extracts derived from“Fragrant Cloud”rose petals(stages4 and5combined)and of partially purified recombinant RhAAT gene product with selected alcohol substrates.Activity with ge-raniol was set as100%.Means and SE s of two replicates.The ex-periments were repeated three times with similar results.Substrate Tested a Crude Extract RhAAT1 Monoterpene alcoholsGeraniol100Ϯ15100Ϯ7.7Citronellol87Ϯ8.860Ϯ3.1Nerol35Ϯ2.815.6Ϯ1.6Linalool22Ϯ0.3 3.6Ϯ0.6 Aromatic alcohols2-Phenylethyl alcohol144Ϯ5.47.9Ϯ0.5Benzyl alcohol35Ϯ0.78.9Ϯ0.5 Aliphatic alcoholscis-3-Hexene1-ol237Ϯ1110.6Ϯ1.21-Hexanol204Ϯ2113.9Ϯ11-Octanol147Ϯ7.734.8Ϯ71-Butanol101Ϯ11 4.2Ϯ0.1Isoamyl alcohol97.4Ϯ14 4.0Ϯ2.01-Decanol50Ϯ0.1 5.1Ϯ0.23-Hexanol28.7Ϯ1.2 4.5Ϯ0.2Ethanol16.5Ϯ1.2 2.4Ϯ0.3a All substrates were tested at a conentration of10m M.Shalit et al.To test for the substrate specificity of the RhAAT1gene product for potential alcoholic substrates and to determine its general catalytic and kinetic parame-ters,we used a simple and sensitive radio-assay as well as gas chromatography-mass spectrometry (GC-MS)analysis for the identification of products (Du-dareva et al.,1998;Shalit et al.,2001).The RhAAT1protein was partially purified (see “Materials andMethods ”),and a number of alcohols were tested as substrates (Table I).RhAAT1was most active with geraniol,catalyzing the production of geranyl acetate from geraniol and acetyl-CoA (Fig.4A).The reaction depended on the presence of enzyme (Fig.4B),on the presence of the alcoholic substrate geraniol (Fig.4C),and acetyl-CoA (Fig.4D).Controls of the same cells lacking a recombinant RhAAT1gene did notpossessFigure parison of the amino acid sequence of the RhAAT1enzyme with closely related sequences (A)and positioning of RhAAT1gene on the phylogenetic map (B).A,RhAAT1(accession no.BQ106456),BEAT (accession no.AF043464),and SAAT (accession no.AF193789)amino acid sequences are compared.Amino acid shaded in black represent identical matches;gray-shaded boxes represent conservative changes.B,Non-rooted phylogenetic tree.Alignments and phylogenetic tree program used was ClustalX (Thompson et al.,1997).Geraniol/Citronellol Acetyltransferase in Rose PetalsAAT activity (Fig.4E).The 2-saturated derivative citronellol was accepted as a substrate at an interme-diate rate (60%as compared with geraniol).Only lowlevels of activity were observed with nerol (16%),a cis isomer of geraniol,and almost no activity (3.6%)was attained with the tertiary monoterpene alcohol linalool.A moderate level of activity was also ob-served with 1-octanol (35%),lesser activity with 1-hexanol (14%),and cis -3-hexen 1-alcohol (10%).Only a slight level of activity was observed when benzyl alcohol,n-butanol,ethanol,and isoamyl alco-hol (9%–2%)were offered as substrates.Interestingly,RhAAT1had low activity (8%)with 2-phenylethyl alcohol compared with geraniol,even though 2-phenylethyl alcohol is the apparent substrate for the formation of 2-phenylethyl acetate,the major vol-atile emitted from “Fragrant Cloud ”flowers,and AAT enzymatic activity catalyzing the formation 2-phenylethyl acetate is higher than geranyl acetate-forming AAT activity in cell-free extracts derived from petals (Table I).Characterization of Kinetic Parameters of the RhAAT1Enzymatic ActivityThe determination of the general catalytic properties and kinetic parameters of the recombinant partially purified RhAAT1enzyme was done utilizing geraniol,citronellol,1-hexanol,and 1-octanol as alcoholic sub-strates and acetyl-CoA as acyl donor.The apparent K m values were:geraniol,0.16m m ;citronellol,0.20m m ;1-hexanol,0.5m m ;and 1-octanol,0.46m m .The K m value for acetyl-CoA was 65m regardless of the alcoholic substrate used.The size of the purified na-tive enzyme,determined by gel filtration chromatog-raphy,was 53kD,similar to the predicted size of the subunit (51.8kD),indicating that the enzyme is active as a monomer,as noted earlier for other plant AATs of the BAHD family (St-Pierre and De Luca,2000).The optimum temperature for catalysis was in the range of 25°C to 35°C.The pH optimum was between pH 7.0and 8.0.The addition of EDTA to the reaction buffer did not affect the activity,indicating a divalent metal-independent catalysis.RhAAT1Expression Is Regulated during Flower DevelopmentWe monitored the level of RhAAT1transcript levels in petals from different developmental stages (1,2,4,and 6)as well as from leaves (Fig.5).No RhAAT1transcripts were detected in leaves and in the early stages of flower development (stage 1).RhAAT1transcript reached maximum level at open flowers (stage 4),and lower transcript was detected at full bloom (stage 6).DISCUSSIONPetals of “Fragrant Cloud ”Roses Display AATsCapable of Synthesizing Many Volatile Acetyl EstersIn previous work,the major constituents of the floral scent of the rose var.“Fragrant Cloud ”wereFigure 4.Identification by GC-MS of the acetate esters product formed in vitro by the product of RhAAT1.Bacterial lysates overex-pressing RhAAT 1were incubated with geraniol and acetyl-CoA in assay buffer as described in “Materials and Methods.”The identifica-tion of the geranyl acetate product was done by matching with the retention time and the mass spectrum of authentic geranyl acetate,and by comparison with the computerized Wiley library.The Kovac index of geranyl acetate also corresponded with that of authentic standard.A,Lysates (derived from cells overexpressing RhAAT1)ϩgeraniol ϩacetyl-CoA.B,Reaction buffer ϩgeraniol ϩacetyl-CoA.C,Lysates derived from cells overexpressing RhAAT1ϩacetyl-CoA.D,Lysates derived from cells overexpressing RhAAT1ϩgeraniol (no acetyl-CoA).E,Control lysates (derived from control E.coli BL21[DE3]p LysS cells,not overexpressing RhAAT1)ϩgeraniol ϩacetyl-CoA.Shalit et al.determined and found to be mostly acetate esters,alcohols,monoterpenes,and sesquiterpenes (Guter-man et al.,2002;Lavid et al.,2002).The emission of high levels of volatile acetate esters suggested that AAT enzymatic activities might be involved in flower scent production as noted before for C.breweri (Dudareva et al.,1998).We tested this hypothesis by measuring AAT activity in crude extracts derived from petals with acetyl-CoA and various alcoholic substrates.The results (Fig.2;Table I)show a signif-icant correlation between the presence of specific acetate esters in the headspace and the ability of petal cell-free extracts to produce these acetate esters in vitro.Nonetheless,the cell-free extracts have the po-tential to acetylate alcohols whose acetate esters are not detected in the headspace of “Fragrant Cloud ”flowers.Thus,the production of acetate esters in rose petals depends not only on the specificity of the AATs present,but also on other factors that may include substrate availability and cell and tissue com-partmentalization of substrates and enzymes.RhAAT1Is an Acetyltransferase with Limited Substrate SpecificityScreening a rose petal EST database for sequences with similarity to known AATs of the BAHD acyl-transferase family of plants (St-Pierre and De Luca,2000)yielded three cDNAs.One of them,RhAAT1,was analyzed in detail in this study and shown to encode an enzyme with high affinity for geraniol that catalyzes the formation of geranyl acetate.The en-zyme also readily accepts citronellol,a 2-saturated derivative of geraniol (approximately 60%the rate obtained with geraniol),but is relatively inefficient (approximately 16%the rate obtained with geraniol)in acetylating nerol,the cis isomer of geraniol.The enzyme had also lower levels of activity with cis -3-hexene 1-alcohol and 2-phenylethyl alcohol (Table I).Therefore,it is likely that cis -3-hexenyl acetate and2-phenylethyl acetate,the two main esters emitted from “Fragrant Cloud ”roses (Fig.2A),are synthe-sized by another enzyme or enzymes.This hypothe-sis is also supported by the observation that the patterns of the changes in cis -3-hexenyl-1-acetate and 2-phenylethyl acetate-forming AAT activity levels over the developmental stages of the flower,al-though similar to each other (Fig.2C),are different from the corresponding pattern of geranyl acetate-forming AAT activity levels (Fig.2B).The maximum for the former occurs at stage 3,whereas the maxi-mum for the latter occurs at stage 4.RhAAT1Is Similar to SAAT and Other Acetyltransferases in the BAHD FamilyThe phylogenetic analysis (Fig.3B)indicates that the enzyme encoded by RhAAT1is most similar to SAAT,an enzyme involved in the production of vol-atile acetate esters in strawberry fruits.Both rose and strawberry belong to the Rosaceae family,and the high sequence similarity (69%)may indicate that the two sequences are descendants from a common gene,diverging after recent gene duplications (Pichersky and Gang,2000).Although SAAT has marked AAT activity with medium-chain alcohol substrates and is less active with short-chain alcohols,the highest ac-tivity of RhAAT1was obtained when the monoter-pene alcohol geraniol was used as a substrate.How-ever,RhAAT1also has marked activity with medium-chain alcohols and some but lesser activity with short-chain alcohols.The potential acetylating activity of SAAT toward the monoterpene alcohols geraniol and citronellol has not been reported.It is also interesting that the K m value of RhAAT1for geraniol,the preferred substrate,is 0.16m m ,whereas it is 0.20,0.45,and 0.5m m for citronellol,1-octanol,and 1-hexanol,respectively.In contrast,the cal-culated K m value of SAAT with 1-octanol and 1-hexanol,the best alcoholic substrates,are much higher (5.7and 8.9m m ,respectively).Regulation of Volatile Emission from Rose Petals at the Enzymatic and Transcriptional LevelEmission of acetate volatiles is very low at stages 1and 2,it became apparent at stage 3of flower devel-opment,and then rapidly increased to a maximum at stage 5.The major acetates emitted are 2-phenyl ethy-lacetate and cis -3-hexenyl acetate.Nevertheless,acetyltransferase activity able to utilize 2-phenylethyl alcohol and cis -3-hexenyl alcohol (the corresponding precursors)is already prominent at stages 1and 2.It could be possible that substrate availability is limit-ing the formation of the corresponding acetates.The levels of the emission of the corresponding alcohol substrates are very low at stages 1and 2and increase at stage 3(M.Shalit and E.Lewinsohn,unpublished data),paralleling the increase in acetate emissions.ItFigure 5.RNA gel-blot analysis of RNA samples derived from “Fra-grant Cloud ”roses.RNA gel-blot analyses of RhAAT1.RNA was extracted from rose cv “Fragrant Cloud ”leaves (L)and from petals at different developmental stages (1,2,4,and 6)and analyzed for RhAAT1expression.Ethidium bromide staining of rRNA is presented (bottom).Geraniol/Citronellol Acetyltransferase in Rose Petalsis also possible that the alcohols and/or the acetates might be formed in the petals at stages1and2but not emitted due to anatomical,developmental,or other physiological constrains.This possibility is un-likely,as indicated by studies in which rose petals were solvent(methyl-tert-butyl ether),extracted,and found to be devoid of these alcohols and their ace-tates(data not shown).Nevertheless,other processes, such as tissue or subcellular compartmentization of the enzymes and substrates,can explain these obser-vations.Moreover,maximal emission of these com-pounds occurs up to stage5,whereas the maximal recorded enzyme activity is at stage3.Therefore,it seems that the nonoptimal levels of enzyme activity can account for the emissions observed.It can also be possible that due to low turnover rates of the ester products,they are maximally emitted at a stage in which enzyme activities are already declining.Con-cerning the emission of geranyl and citronellyl ace-tates,the maximal levels of the measured AAT en-zyme activities also do not correlate exactly with the highest levels of volatile emission.It could be that the high geraniol-and citronellol-specific AAT activities observed in cell-free extracts in stages1and2are due to other AAT enzyme activities compartmentized from their substrates.This is corroborated by the RhAAT1expression analyses,which indicate an ap-parent lack of RhAAT1transcript at stages1and2. Therefore,we assume that other acetyltransferases that could use geraniol and citronellol as potential substrates might yield substantial activity measure-ments in stages1and2.Moreover,two additional cDNAs with sequences similar to genes of the BAHD family have been identified in the“Fragrant Cloud”rose EST database,but have not yet been fully char-acterized.In accordance,it seems that additional AAT genes could be involved in formation of volatile esters emitted from the flowers.CONCLUSIONVolatile acetate esters are very important contrib-utors to the aroma of many plants,including com-mercially important crops.Only a few genes encod-ing enzymes involved in volatile ester formation have been identified and characterized.We have de-scribed the identification and characterization of the gene that codes for RhAAT1,an enzyme able to catalyze the formation of volatile esters in a major ornamental crop.The rose RhAAT1is currently the only known AAT that can utilize the monoterpene geraniol as substrate to generate geranyl acetate,a compound with a fruity rose note reminiscent of pear (Pyrus communis)and slightly of lavender that occurs in the scent of many plants(Bauer et al.,2001).The availability of RhAAT1and similar AAT genes that encode enzymes for the formation of fragrances will allow a better understanding of the factors that in-fluence and limit the biogeneration of scent com-pounds.This information can be used to generate crops with improved or modified fragrance using the modern biotechnological tools available(Galili et al., 2002).MATERIALS AND METHODSPlant MaterialFlowers of rose(Rosa hybrida)cv“Fragrant Cloud”were harvested from plants grown in a greenhouse in the Newe-Ya’ar Research Center,Israel, under controlled conditions(Lavid et al.,2002).Chemicals and RadiochemicalsAll chemicals and radiochemicals were purchased from Sigma(St.Louis) unless otherwise noted.Headspace Volatile CollectionIntact individual rose flowers,still attached to the bush,were enclosed in a1-L glass container with the appropriate openings,and headspace was trapped for24h at25°C using a method modified from Raguso and Pichersky(1995),utilizing a Porapak Q80/100(Waters Corp.,Milford,MA) polydivinylbenzene filter.Photon fluence was22Ϯ2E mϪ2sϪ1as deter-mined by an LI-188B integrating quantum radiometer/photometer(LI-COR, Lincoln,NE).The photoperiod was15h,and the starting of the sampling was initiated4h after the lights were turned on.Volatiles were eluted utilizing10mL of HPLC-grade hexane containing100g of ethylmyristate as an internal standard and evaporated to0.5mL.One microliter of each sample was analyzed by GC-MS(Lavid et al.,2002).GC-MS AnalysisThe volatile compounds collected from the headspace were analyzed on an Hewlett-Packard-GCD apparatus equipped with an HP-5(30mϫ0.25 mm)fused-silica capillary column.Helium(1mL minϪ1)was used as a carrier gas.The injector temperature was250°C,set for splitless injection. The oven was set to50°C for1min,and then the temperature was increased to200°C at a rate of4°C minϪ1.The detector temperature was280°C.Mass range was recorded from45to450mass-to-charge ratio,with electron energy of70eV.Identification of the main components was done by comparison of mass spectra and retention time data with those of authentic samples and supplemented with a Wiley GC-MS library(Lewinsohn et al., 2001;Shalit et al.,2001).Quantification of the compounds was performed by utilizing the total mass ions detected and compared with the internal standard.Cloning of Rose AAT GeneRhAAT1was identified in the“Fragrant Cloud”petal EST database (Guterman et al.,2002)by homology search(BLAST)with other AATs. RhAAT1was subcloned into a T7-dependent expression vector pET(11a)by PCR with the appropriate oligonucleotides according to the manufacturer’s instructions as previously described(Wang and Pichersky,1999).RNA Extraction and AnalysisTotal RNA was extracted from petals and leaves as previously described (Manning,1991).RNA samples(10g)were fractionated in a1%(w/v) agarose gel containing formaldehyde and blotted onto Hybond Nϩmem-branes(Amersham,Buckinghamshire,UK).The blots were hybridized in a solution containing0.26m Na2PO4,7%(w/v)SDS,1m m EDTA,and1% (w/v)bovine serum albumin at60°C with32P-labeled RhAAT1cDNA probe (Redprime,Amersham).The membranes were washed twice in2ϫSSC and 0.1%(w/v)SDS at60°C for20min each and exposed to x-ray film(Fuji, Tokyo;Lavid et al.,2002).Shalit et al.。
植物氨基酸分解代谢产生乙酰辅酶a
植物氨基酸分解代谢产生乙酰辅酶a
植物细胞中的氨基酸分解代谢是一种重要的代谢途径，能够提供植物细胞所需的能量、有机酸以及氮源。
在这一过程中，通过氨基酸的脱羧反应和氨基转移反应，可以产生一系
列代谢产物和中间产物，其中就包括了乙酰辅酶A（acetyl-CoA）。
乙酰辅酶A是一种重要的代谢分子，它是三羧酸循环、碳水化合物代谢、脂肪酸合成
以及许多其他代谢途径中的中间体。
在植物细胞中，乙酰辅酶A的来源主要有两种途径，
一种是从葡萄糖代谢中产生的，另一种则是通过氨基酸分解代谢产生的。
氨基酸的脱羧反应一般由氨基酸脱羧酶（amino acid decarboxylase）催化，该反应
产生CO2和酰基胺（amino acid）。
酰基胺在此后进行氨基转移反应，由氨基转移酶（aminotransferase）催化，反应产生对应的酮酸（keto acid）和谷氨酰胺（glutamine）。
在氨基酸分解的过程中，酮酸可以通过另一种脱羧反应产生乙酰辅酶A。
乙酰辅酶A在植物细胞中有着多种重要的功能。
在糖酵解中，乙酰辅酶A参与三羧酸
循环的前一步反应，产生能量和CO2。
在植物脂肪酸合成中，乙酰辅酶A被羧化成丙酮酸后，在合成途径中不断被链延伸，最终形成脂质分子。
此外，乙酰辅酶A还参与着许多其
他代谢途径的进行，如胆固醇合成和非生物诱导的防御代谢等。
总的来说，植物氨基酸分解代谢产生的乙酰辅酶A，是植物细胞代谢活性的重要中间
体之一，对于植物细胞能量代谢和生物合成过程具有重要作用。
乙酰辅酶a的来源与去路
乙酰辅酶a的来源与去路
乙酰辅酶A是能量物质代谢的重要中间代谢产物，是体内能量物质代谢的关键物质。
乙酰辅酶A可通过糖、脂肪和氨基酸的分解代谢产生，也可合成脂肪酸、胆固醇、酮并参与乙酰化反应。
来源：糖的有氧氧化；葡萄糖丙酮酸乙酰辅酶A；脂肪酸的合成β-氧化；脂肪酸酰基辅酶A乙酰辅酶A参与某些氨基酸的分解代谢；酮的氧化分解。
β-羟基丁酸乙酰乙酸辅酶a。
去路：进入三羧酸循环并完全氧化；在肝脏中合成酮；合成脂肪酸和胆固醇；参与乙酰化反应。
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
乙酰乙酰辅酶a结构式
乙酰乙酰辅酶A（CoA）是一种有机磷脂，它是由一种胰脂肪醇和腺苷酸乙酰胆碱（ACP）经过细胞内转移酶作用结合而成的。
它的结构式如下：
结构图
乙酰乙酰辅酶A是细胞能量代谢的中间代谢物形成的巨胞体。
它可以与与它结合的转肽酶以及脂肪酸合成过程中的一些重要反应物结合，将这些重要反应物转化为具有能量存储功能的ATP分子，以便细胞使用。
乙酰乙酰辅酶A也可以与脂肪酸二酯分解酶结合，并参与脂肪酸的氧化分解。
乙酰乙酰辅酶A在代谢系统中发挥着重要作用，它可以辅助细胞将外部能量转化为能量状态。
它可以作为细胞能量反应的调节剂，可以在细胞内和细胞外参与各种重要的生物反应，如脂肪酸的氧化分解，糖的分解，脂的分解，酮的形成等。
乙酰乙酰辅酶也可以用作检测血液糖水平的指标曲线，用以判定糖尿病患者状态，也可以在血液护理中用作检测代谢性疾病的标志物，以提供有效的治疗方法。
因此，乙酰乙酰辅酶A在能源代谢中发挥着重要的作用，对生理和病理的研究也有重要意义，因此，乙酰乙酰辅酶A的研究是至关重要的。