Biosynthesis of dicarboxylic acids by carbon dioxide fixation; isolation and properties of an enzyme from pigeon liver catalyzing the reversible oxidative decarboxylation of 1-malic acid.-猫眼课题宝

DOI：

10.1016/s0021-9258(18)55935-4

发表时间：

1948-07

期刊：

The Journal of biological chemistry

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

S. Ochoa;A. Mehler;A. Kornberg

二氧化二羧酸的生物合成固定VI.苹果酸和约克的分离，通过碳酸 * 考夫曼学院由SEVER 0 OCHOA New SEYMOUR纽约）（来自药物学系，（1951年4月2日接收于纽约大学），供出版，通过苹果酸的还原性羧化来生物合成L-苹果酸已经在前面描述过（1）。在鸽肝”苹果酸”酶和葡萄糖磷酸脱氢酶存在下，由于TPN连接的歧化，容易形成苹果酸葡萄糖-6-磷酸+丙酮酸+ CO2（TPN，Mn++）+6-磷酸葡萄糖酸+n-苹果酸。酶反应-反应混合物如下：葡萄糖B-磷酸盐，10 mM;丙酮酸盐，10 mM; NaHCO 3，15 mM; MnO 2，0.2 mM;三磷酸吡啶核苷酸（TPN），0.027 mM;葡萄糖磷酸脱氢酶，1250单位;鸽肝”malie”酶（2），8000单位。将体积补足至101 cc。用75%二氧化碳和25%氮气的气体混合物平衡溶液，使pH值达到7.0。将混合物在25”下振荡孵育4小时。在1.0 cc.如已经描述的（1），将上述反应混合物等分。在4小时内，在100 cc中产生了3.15 mM的磷酸葡萄糖酸盐。的主要反应混合物。此时，用2.7cc将溶液调至pH 5.5。IN硫酸，在100 ° C下加热8分钟，然后在冰中冷却。通过离心除去蛋白质沉淀。由于所用的葡萄糖磷酸脱氢酶粗品制剂被脱氢酶污染，因此预期反应会形成富马酸盐和n-苹果酸盐的混合物。由美国公共卫生署、美国癌症协会（由国家研究理事会增长委员会推荐）、海军研究办公室和美国氰胺公司Lederle实验室分部的赠款资助。313这是CC BY许可下的开放获取文章。314二羧酸的生物合成。VI.在存在蔗糖酶的情况下，用阿拉伯乳杆菌”苹果酸”酶的两种二羧酸的总和（1）表明，2.56 mM（形成的磷酸葡萄糖酸的81%）（参见(1))已经生产出来了。因此，25%的丙酮酸被转化为二羧酸。L-Ma& A&的分离--将不完全无蛋白质的溶液用钨酸完全脱蛋白质并过滤。用硫酸将含有1.95mM富马酸+L-苹果酸（或如果已建立酶平衡，则约1.45mM正苹果酸）的滤液的等分试样调至pH 1.0，并用乙醚连续萃取100小时。蒸发乙醚，残留物溶于水中，体积补足至25毫升。该溶液含有0.92 mM的n-苹果酸（用无酶的鸽肝”苹果酸”酶（3）测定）和1.09 mM的N-苹果酸（用乳酸脱氢酶（3）测定）。10毫升将上述溶液的等分试样（~ 0.37 μ g的n-苹果酸）在pH8.0下冻干，并如论文V（4）中所述在硅胶柱上进行色谱分析。0.17获得了1 mM的苹果酸。辛可宁盐-在含有0.1mM苹果酸的流出液的等分试样上制备辛可宁盐（5）。18.4 mg.的粗盐。经过一次重结晶后...

BIOSYNTHESIS OF DICARBOXYLIC ACIDS DIOXIDE FIXATION VI. ISOLATION OF MALIC AND York, BY CARBON ACID* KAUFMAN College BY SEVER0 OCHOA New SEYMOUR New York)(From the Department of Pharmacology,(Received New York University April 2, 1951) of Medicine, for publication, The biosynthesis of L-malic acid by reductive carboxylation of pyruvic acid has been previously described (1). Malate is readily formed, in the presence of pigeon liver" malic" enzyme and glucose phosphate dehydrogenase, as a result of the TPN-linked dismutation Glucose-6-phosphate+ pyruvate+ CO2 (TPN, Mn++)+ 6-phosphogluconate+ n-malate While in the previous work L-malic acid had been identified and determined by enzymatic methods (1)) it has now been isolated and identified chemically. Enzyme Reaction-The reaction mixture was as follows: glucoseB-phosphate, 10 mM; pyruvate, 10 mM; NaHC03, 15 mM; MnC&, 0.2 mM; triphosphopyridine nucleotide (TPN), 0.027 mM; glucose phosphate dehydrogenase, 1250 units; pigeon liver" malie" enzyme (2)) 8000 units. The volume was made up to 101 cc. with water and the solution equilibrated with a gas mixture containing 75 per cent carbon dioxide and 25 per cent nitrogen, bringing the pH to 7.0. The mixture was incubated with shaking at 25" for 4 hours. The course of the reaction was followed manometrically, on a 1.0 cc. aliquot of the above reaction mixture, as already described (1). In 4 hours, 3.15 mM of phosphogluconate were produced in 100 cc. of the main reaction mixture. At this time the solution was brought to pH 5.5 with 2.7 cc. of 10.0 N sulfuric acid, heated at 100" for 8 minutes, and then cooled in ice. The protein precipitate was removed by centrifugation. Since the crude preparation of glucose phosphate dehydrogenase used was contaminated with fumarase, the formation of a mixture of fumarate and n-malate was expected as a result of the reaction. Determination of* Aided by grants from the United States Public Health Service, the American Cancer Society (recommended by the Committee on Growth of the National Research Council), the Office of Naval Research, and the Lederle Laboratories Division, American Cyanamid Company. 313 This is an Open Access article under the CC BY license. 314 BIOSYNTHESIS OF DICARBOXYLIC ACIDS. VI the sum of the two dicarboxylic acids with Lactobacillus arabinosus" malic" enzyme in the presence of fumarase (1) showed that 2.56 mM (81 per cent of the phosphogluconate formed (cf.(1)) had been produced. Thus, 25 per cent of the pyruvate was converted to dicarboxylic acids. Isolation of L-Ma& A&--The solution, which was not quite proteinfree, was completely deproteinized with tungstic acid and filtered. An aliquot of the filtrate containing 1.95 mM of fumaric+ L-malic acid (or about 1.45 mM of n-malic acid if fumarase equilibrium had been established) was brought to pH 1.0 with sulfuric acid and extracted continuously with ether for 100 hours. The ether was evaporated, the residue taken up in water, and the volume made up to 25 cc. This solution contained 0.92 mM of n-malic acid, as determined spectrophotometrically with fumarase-free, pigeon liver" malic" enzyme (3), and 1.09 mu of pyruvic acid, as determined with lactic dehydrogenase (3). A 10 cc. aliquot of the above solution (-0.37 mu of n-malic acid) was lyophilized at pH 8.0 and chromatographed on a silica gel column as described in Paper V (4). 0.17 mM of malic acid was obtained. Cinchonine Salt-The cinchonine salt was prepared (5) on an aliquot of the effluent containing 0.1 mM of malic acid. 18.4 mg. of the crude salt were obtained. After one recrystallization from …

Biosynthesis of dicarboxylic acids by carbon dioxide fixation; isolation and properties of an enzyme from pigeon liver catalyzing the reversible oxidative decarboxylation of 1-malic acid.

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