大鼠微粒体环氧化物水解酶在大肠杆菌中的表达。对催化作用至关重要的组氨酸残基的鉴定。

Expression of rat microsomal epoxide hydrolase in Escherichia coli. Identification of a histidyl residue essential for catalysis.

作者信息

Bell P A, Kasper C B

机构信息

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.

出版信息

J Biol Chem. 1993 Jul 5;268(19):14011-7.

PMID:8314768

Abstract

The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1. Recombinant epoxide hydrolase was found to represent 4-9% of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo[a]pyrene 4,5-oxide with specific activities of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively. Previous work implicated a histidyl residue at or near the active site of the enzyme (DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 253, 2932-2939). Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence of 14 conserved histidyl residues. To investigate the role of these residues in epoxide hydrolysis, site-specific mutants were generated and expressed in E. coli. Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L, H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo[a]pyrene 4,5-oxide with specific activities between 115 and 830 nmol min-1 mg-1. Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1, which is at least 150-fold less than the activity of the wild type enzyme. A Vm versus pH profile for the recombinant wild type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4. The group with a pKa of 5.8 is preferentially unprotonated, while the other two groups are preferentially protonated for catalysis. We propose that histidine 431 corresponds to the group with a pKa of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues. Thus, the data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s), perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond.

摘要

将含有大鼠微粒体环氧化物水解酶（EC 3.3.2.3）完整编码区的cDNA克隆到表达/分泌载体pIN-III-OmpA3中，并在大肠杆菌TG1菌株中表达。发现重组环氧化物水解酶占细菌总蛋白的4 - 9%，并催化氧化苯乙烯和苯并[a]芘4,5-氧化物的水解，比活性分别为421和734 nmol·min⁻¹·mg环氧化物水解酶⁻¹。先前的研究表明该酶活性位点或其附近存在一个组氨酸残基（DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 253, 2932 - 2939）。大鼠、人及兔环氧化物水解酶氨基酸序列的比较显示存在14个保守的组氨酸残基。为研究这些残基在环氧化物水解中的作用，构建了位点特异性突变体并在大肠杆菌中表达。突变体H64L、H82L、H115N、H126N、H129L、H148N、H170L、H176L、H242L、H247L、H301L、H385L、K386M - H387L、δ385 - 391和H407L催化苯并[a]芘4,5-氧化物的水解，比活性在115至830 nmol·min⁻¹·mg⁻¹之间。发现突变体H431L、H431N和H431R的活性均<5 nmol·min⁻¹·mg⁻¹，这比野生型酶的活性至少低150倍。重组野生型环氧化物水解酶的Vm对pH曲线显示最适pH范围较宽，为6.5至8.5，且存在三个可电离基团，其pKa值分别为5.8±0.2、9.2±0.1和9.7±0.4。pKa为5.8的基团优先处于未质子化状态，而另外两个基团优先质子化以进行催化。我们推测组氨酸431对应于pKa为5.8的基团，而其他pKa值为9.2和9.7的基团可能代表赖氨酸、半胱氨酸或酪氨酸残基。因此，这些数据与一个模型相符，即His - 431作为一般碱从水中夺取一个质子，而另一个残基（可能是赖氨酸）作为一般酸使碳 - 氧键断裂时形成的醇盐阴离子质子化。