3-Hydroxy-3-methylglutaryl coenzyme A lyase: affinity labeling of the Pseudomonas mevalonii enzyme and assignment of cysteine-237 to the active site.
3-Hydroxy-3-methylglutaryl coenzyme A lyase: affinity labeling of the Pseudomonas mevalonii enzyme and assignment of cysteine-237 to the active site.
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3-羟基-3-甲基戊二酰辅酶 A 裂解酶：对 Pseudomonas mevalonii 酶进行亲和标记，并将半胱氨酸 237 分配到活性位点。
DOI：
10.1021/bi00144a026
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Miziorko,HM
中科院分区：
文献类型：
--
作者：
Hruz,PW;Narasimhan,C;Miziorko,HM
修订稿于 1992 年 5 月 5 日收到摘要：Pseudomonas mevalonii3-羟基-3-甲基戊二酰辅酶 A (HMG-CoA) 裂合酶被反应性底物类似物 2-丁酰基-CoA 不可逆地灭活。酶失活遵循伪一级动力学，在 23 C、pH 7.2 下，K\= 65 µ 和 0.073 min-1 的极限 k−1 是可饱和的。通过竞争性抑制剂 3-羟基戊二酰辅酶 A 提供防止失活的保护。用 [1-14C]-2-丁酰辅酶 A 标记细菌酶表明失活与共价一致。掺入抑制剂，每个位点的化学计量为 0.65，禽 HMG-CoA 裂解酶也会被每个位点的化学计量为 0.9 的 2-丁酰辅酶 A 灭活。2-丁酰辅酶 A 与硫醇（如二硫苏糖醇）一起孵育也会导致在 310 nm 处形成 UV 吸光度峰。 310 nm 处的 UV 吸光度峰表明 2-丁酰基-CoA 修饰了 HMG-CoA 裂解酶中的半胱氨酸残基。亲和标记蛋白的胰蛋白酶消化和反相 HPLC 揭示了该肽的单个放射性标记肽和较小的胰凝乳蛋白酶肽，表明放射性标记残基包含在 cDNA 推导的序列 GGXPY 内。 P. mevalonii HMG-CoA 裂解酶的序列 [Anderson, D. H., & Rodwell, VW (1989) J. Bacterio!. 171, 6468-6472] 证实位置 237 处的半胱氨酸是修饰位点。这些数据代表了 HMG-CoA 裂解酶中活性位点残基的首次鉴定。 (HMG-CoA) 1 裂解酶 (EC 4.1. 3.4) 在生酮 HMG-CoA 循环的最后一步催化 HMG-CoA 裂解为乙酰乙酸和乙酰-CoA。除了在生酮作用中发挥重要作用外（Robinson & Williamson，1980），该酶还在氨基酸亮氨酸遗传性缺乏的分解代谢途径中发挥作用。据报道，这种酶在禁食或感染后会导致严重的代谢性酸中毒（Gibson 等人，1988）；尽管 HMG-CoA 裂解酶很重要，但直到最近才从包括猪心脏在内的许多真核来源中部分纯化出这种酶的结构信息（Bachawat 等人，1991）。 1955）和牛肝脏（Stegink & Coon，1968），并且从禽类肝脏中获得了均质酶（Kramer & Miziorko，1980）。该酶也从原核生物梅瓦隆假单胞菌中部分纯化（Scher & Rodwell，1989）Anderson 和 Rodwell（1989）报道了梅瓦隆假单胞菌的 cDNA 推导的一级序列。 Mitchell 等人（1991）最近报道了禽类和人类 HMG-CoA 裂解酶的推导的一级序列。裂解酶反应被认为是从 3- 中提取质子的逆克莱森缩合反应。
Revised Manuscript Received May 5, 1992 abstract: Pseudomonas mevalonii3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase is irreversibly inactivated by the reactive substrate analog 2-butynoyl-CoA. Enzyme inactivation, which follows pseudo-first-order kinetics, is saturable with a K\= 65 µ and a limiting k¡„act of 0.073 min-1 at 23 C, pH 7.2. Protection against inactivation is afforded by the competitive inhibitor 3-hydroxyglutaryl-CoA. Labeling of the bacterial enzyme with [1-14C]-2-butynoyl-CoA demonstrates that inactivation coincides with covalent incorporation of inhibitor, with an observed stoichiometry of modification of 0.65 per site. Avian HMG-CoA lyase is also irreversibly inactivated by 2-butynoyl-CoA with a stoichiometry of modification of 0.9 per site. Incubation of 2-butynoyl-CoA with mercaptans such as dithiothreitol results in the formation of a UV absorbance peak at 310 nm. Enzyme inactivation is also accompanied by the development of a UV absorbance peak at 310 nm indicating that 2-butynoyl-CoA modifies a cysteine residue in HMG-CoA lyase. Tryptic digestion and reverse-phase HPLC of the affinity-labeled protein reveal a single radiolabeled peptide. Isolation and sequence analysis of this peptide and a smaller chymotryptic peptide indicate that the radiolabeled residue is contained within the sequence GGXPY. Mapping of this peptide within the cDNA-deduced sequence of P. mevalonii HMG-CoA lyase [Anderson, D. H., & Rodwell, VW (1989) J. Bacterio!. 171, 6468-6472] confirms that a cysteine at position 237is the site of modification. These data represent the first identification of an active-site residue in HMG-CoA lyase.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 1 lyase (EC 4.1. 3.4) catalyzes the cleavage of HMG-CoA into acetoacetate and acetyl-CoA in the final step of the ketogenic HMG-CoA cycle. In additionto its vital role in ketogenesis (Robinson & Williamson, 1980), the enzyme also functions in the catabolic pathway of the amino acid leucine. Inherited deficiency of this enzyme has been reported to cause severe metabolic acidosis following periods of fasting or infection (Gibson et al., 1988); this disease has proven fatal in a number of cases (Ozand et al., 1991). Despite the importance of HMG-CoA lyase, only recently has structural information become available for this enzyme. HMG-CoA lyase has been partially purified from a number of eukaryotic sources including pig heart (Bachawat et al., 1955) and bovine liver (Stegink & Coon, 1968), and homogeneous enzyme has been obtained from avian liver (Kramer & Miziorko, 1980). The enzyme has also been partially purified from the prokaryote Pseudo-monas mevalonii (Scher & Rodwell, 1989). Anderson and Rodwell (1989) have reported the cDNA-deduced primary sequence of the P. mevalonii enzyme, and Mitchell etal.(1991) have recently reported thededuced primary sequences of both avian and human HMG-CoA lyases. The lyase reaction is believed to be a retro-Claisen condensation with the abstraction of a proton from the3-