Sundberg K, Johansson A S, Stenberg G, Widersten M, Seidel A, Mannervik B, Jernström B
Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Stockholm, Sweden.
Carcinogenesis. 1998 Mar;19(3):433-6. doi: 10.1093/carcin/19.3.433.
Previous studies have identified allelic variants of the human glutathione transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (kcat/Km) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti- and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest kcat/Km value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of kcat/Km. With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased kcat/Km values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionylation. In conclusion, the present study indicates that individuals who are homozygous for the allele GSTP1* B (coding for GSTP1-1/V-105) display a higher susceptibility to malignancy because of other factors than a decreased catalytic efficiency of GSTP1-1/V-105 in the detoxication of carcinogenic diol epoxides of benzo[a]pyrene or structurally related PAH.
以往的研究已鉴定出人类谷胱甘肽转移酶（GST）Pi基因的等位基因变体，并表明在第105位分别编码异亮氨酸（GSTP1-1/I-105）或缬氨酸（GSTP1-1/V-105）的两种不同蛋白质，与模型底物的催化活性存在显著差异。此外，最近的流行病学研究表明，这些等位基因变体表达不同的个体，在某些器官中形成肿瘤的易感性也不同，包括那些多环芳烃（PAH）可能作为病因的器官。在本研究中，测定了这些GSTP1-1变体与多种立体异构的湾区二醇环氧化物的催化效率（kcat/Km），这些二醇环氧化物是已知的PAH的最终诱变和致癌代谢物，包括来自芘、苯并[a]芘和二苯并[a,h]蒽的代谢物。此外，已构建并表征了第105位氨基酸残基为丙氨酸（GSTP1-1/A-105）或色氨酸（GSTP1-1/W-105）的GSTP1-1突变体。发现GSTP1-1/V-105在与PAH的大分子二醇环氧化物的结合反应中比GSTP1-1/I-105更具活性，对苄基环氧乙烷基碳上具有R-绝对构型的反式和顺式二醇环氧化物对映体的活性高达3倍。比较这四种酶变体，GSTP1-1/A-105通常表现出最高的kcat/Km值，而GSTP1-1/W-105与反式二醇环氧化物反应时表现出最低的值。观察到第105位氨基酸残基占据的体积与kcat/Km值之间存在密切相关性。对于顺式二醇环氧化物，在丙氨酸、缬氨酸和异亮氨酸之间观察到这种相关性，而色氨酸与kcat/Km值增加有关。与天然存在的等位基因变体GSTP1-1/I-105和GSTP1-1/V-105相比，第105位异亮氨酸被丙氨酸或色氨酸的突变替代并未改变GSTP1-1变体的对映选择性。由于第105位的氨基酸构成底物结合位点（H-位点）的一部分，预期增加体积的影响会导致二醇环氧化物的进入受限以及两种反应物的正确排列以实现有效的谷胱甘肽化。总之，本研究表明，对于等位基因GSTP1*B（编码GSTP1-1/V-105）纯合的个体，由于除GSTP1-1/V-105在苯并[a]芘或结构相关PAH的致癌二醇环氧化物解毒中催化效率降低之外的其他因素，表现出更高的恶性肿瘤易感性。
