Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A.
Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A.
复制标题
3-氯丙酰辅酶 A 对 3-羟基-3-甲基戊二酰辅酶 A 合酶的活性位点定向抑制。
DOI：
10.1021/bi00334a015
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Behnke,CE
中科院分区：
文献类型：
--
作者：
Miziorko,HM;Behnke,CE
方法3-氯丙酰辅酶A的合成。3-氯-丙酰基-CoA通过Simon & Shemin（1953）的方法制备。在0 ℃下，将5倍摩尔过量的再蒸馏3-氯丙酰氯以小份加入CoASH（锂盐）在0.4MKHCG3中的溶液中，pH7.0-7.5.当用硝普钠试剂测试时，溶液给出阴性响应，表明不存在游离巯基后，将溶液的pH降低至3.5。然后用乙醚萃取溶液。将含有反应产物的水层调至pH 4.5-5.0。3-氯丙酰-CoA通过Whatman DE 52纤维素上的柱色谱法纯化，使用含有3 mM HCl的20-200 mM LiCl线性梯度，随后在Sephadex G10柱上层析。将3-氯丙酰-CoA在-20 ℃下以溶液或冻干粉的形式储存。通过反相HPLC [LiChrospherRP-18; 100 mM四丁基磷酸铵（pH 5.5）/甲醇（45：55）]评估产物的纯度。[11 C]-3-氯丙酰基-CoA的合成。3-将氯[1 - 14 C]丙酸溶解在25倍摩尔过量的草酰氯中，并将所得溶液在50 ℃下孵育30分钟以形成酰氯（Kass和Brock，1969）。未反应的草酰氯通过使用干燥N2流挥发。将产物溶解在乙醚中，并将最后痕量的未反应试剂与溶剂一起在干燥N2下沿着蒸发。将放射性产物的等分试样以及真实的3-氯丙酰氯样品与中性羟胺反应。所得异羟肟酸盐在薄层色谱（纤维素;水饱和的1-丁醇）上迁移相同。[14 C]-3-氯丙酰1-CoA是按照上述步骤使用14 C标记的酰氯制备的。经反相高效液相色谱分析，DEAE-纤维素纯化产物与未标记物质共迁移。3-氯丙酰基-[3 '-氨基] CoA的合成。将4.0-mL反应混合物在37 ℃下孵育1小时，所述反应混合物在40 mM Tris-HCl（pH 8.2）中含有10 µ脱磷酸-CoA、98 µ [-32]（0.16 Ci/mol）和60毫单位脱磷酸-CoA激酶，并含有30 mM MgCl 2和1 mM DTT。然后将另外15毫单位的酶加入到反应混合物中，继续孵育总共4.8小时。通过冷冻混合物终止反应。产物通过柱色谱法在DEAE-纤维素（1.5 X 25 cm）上使用含有3 mM HCl的10-200 mM LiCl线性梯度纯化。监测馏分的A260和32 P放射性。合并含[32 P] CoASH的级分（通过反相HPLC验证），并使用旋转蒸发器浓缩。将残余物溶于冷甲醇中，并通过加入4体积的冷丙酮沉淀。将干燥的产物储存在-20 ℃。如合成未标记的3-氯丙酰-CoA所述，通过使[3 ′-32 P] CoA与3-氯丙酰氯反应来制备3-氯丙酰-[3 ′-32 P] CoA，不同之处在于使用0.13 M Li 2CO 3代替KHCO 3缓冲液。3-氯丙酰-[32 P] CoA通过重复沉淀纯化，
MethodsSynthesis of 3-Chloropropionyl Coenzyme A. 3-Chloro-propionyl-CoA was prepared by the method of Simon & Shemin (1953). A 5-fold molar excess of redistilled 3-chloropropionyl chloride was added in small portions to a solution of CoASH (lithium salt) in 0.4 M KHCG3, pH7.0-7.5, at 0 C. After the solution gave a negative response when tested with nitroprusside reagent, indicating the absence of free sulfhydryl, the pH of the solution was lowered to 3.5. The solution was then extracted with diethyl ether. The aqueous layer, containing the reaction product, was brought to pH 4.5-5.0. 3-Chloropropionyl-CoA was purified by col-umn chromatography on Whatman DE52 cellulose, using a 20-200 mM LiCl linear gradient containing 3 mM HC1, followed by desalting on a Sephadex G10 column. The 3-chloropropionyl-CoA was stored at-20 C insolution or as a lyophilized powder. The purityof the product was assessed by reverse-phase HPLC [LiChrospherRP-18; 100 mM tet-rabutylammonium phosphate (pH 5.5)/methanol (45: 55)]. Synthesis of [uC]-3-Chloropropionyl-CoA. 3-Chloro [l-14C] propionic acid was dissolved in a 25-fold molar excess of oxalyl chloride, and the resulting solution was incubated for 30 min at 50 C to form the acyl chloride (Kass & Brock, 1969). Unreacted oxalyl chloride was volatilized by using a stream of dry N2. The product was dissolved in diethyl ether, and the last traces of unreacted reagent were evaporated along with solvent underdry N2. An aliquot of the radioactive product, as well as a sample of authentic 3-chloropropionyl chloride, was reacted with neutral hydroxylamine. The re-sulting hydroxamates migrated identically upon thin-layer chromatography (cellulose; water-saturated 1-butanol).[14C]-3-Chloropropiony 1-CoA was prepared by using the 14C-labeled acylchloride in the procedure describedabove. The DEAE-cellulose-purified product comigrated with unla-beled material upon reverse-phase HPLC. Synthesis of 3-Chloropropionyl-[3'-nP] CoA. A 4.0-mL reaction mixture containing 10 µ of dephospho-CoA, 98 µ of [-32](0.16 Ci/mol), and 60 milliunits of de-phospho-CoA kinase in 40 mM Tris-HCl, pH 8.2, with 30 mM MgCl2 and 1 mM DTT was incubated at 37 C for 1 h. An additional 15 milliunits of enzyme was then added to the reaction mixture, and incubation was continued for a total of 4.8 h. The reaction was terminated by freezing the mixture. The product was purifed by column chromatography on DEAE-cellulose (1.5 X 25 cm) using a 10-200 mM LiCl linear gradient containing 3 mM HC1. Fractions were monitored for A260 and 32P radioactivity. The [32P] CoASH-containing fractions (verified byreverse-phase HPLC) were pooled and concentrated by using a rotary evaporator. The residue was dissolved in cold methanol and precipitated by addition of 4 volumes of cold acetone. The dried product was stored at-20 C.3-Chloropropionyl-[3'-32P] CoA was prepared by reacting [3'-32P] CoA with 3-chloropropionylchloride, as described for the synthesis of unlabeled 3-chloropropionyl-CoA, except using 0.13 M Li2C03 instead of the KHC03 buffer. The 3-chloropropionyl-[32P] CoA was purified by repeated precipi-