Kanaani J, Maltby D, Focia P, Wang C C
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Biochemistry. 1995 Nov 21;34(46):14987-96. doi: 10.1021/bi00046a005.
Labeling of human and schistosomal hypoxanthine-guanine phosphoribosyltransferases (HGPRTases) with GMP-2',3'-dialdehyde (ox-GMP) results in nearly complete inactivation of the enzymes. Digestion of the [3H]ox-GMP-modified HGPRTases with trypsin followed by high-performance liquid chromatographic fractionation, partial amino acid sequencing, and mass spectral analysis of the labeled peptides revealed that four peptides from each of the two HGPRTases were labeled with ox-GMP. The conclusion from these studies indicates that two segments of the human enzyme protein, Ser 4-Arg 47 and Ser 91-Arg 100, and one region in the schistosomal enzyme, Gly 95-Lys 133, were labeled by ox-GMP. Since the ox-GMP labeling of human HGPRTase was effectively blocked by either GMP or PRibPP, whereas that of schistosomal HGPRTase was inhibited only by GMP [Kanaaneh, J., Craig, S. P., III, & Wang, C. C. (1994) Eur. J. Biochem. 223, 595-601], the two labeled peptides in human enzyme may be involved in binding to both GMP and PRibPP while the one peptide in schistosomal enzyme may be implicated only in GMP binding. We have also confirmed a previous observation [Keough, D. T., Emmerson, B. T., & de Jersey, J. (1991) Biochim. Biophys. Acta 1096, 95-100] that carboxymethylation of Cys 22 in the human HGPRTase by iodoacetate was inhibited by PRibPP. We also demonstrated that the carboxymethylation of Cys 25 in schistosomal HGPRTase by iodoacetate was specifically blocked by PRibPP. Apparently, the N-terminal regions in both enzymes are involved in PRibPP binding. The fact that ox-GMP labels the N-terminal region in human enzyme but not in schistosomal enzyme and that PRibPP protects against ox-GMP labeling in human enzyme but not in schistosomal enzyme both suggest that the amino-terminal PRibPP-binding site may be in close proximity to the GMP-binding site in human HGPRTase but not in schistosomal HGPRTase. This clear distinction between the active sites of human and schistosomal HGPRTases could be further exploited for potential opportunities for antischistosomal chemotherapy.
用鸟苷-2',3'-二醛（氧化型鸟苷，ox-GMP）标记人和血吸虫次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRTases），几乎可使这些酶完全失活。用胰蛋白酶消化经[³H]ox-GMP修饰的HGPRTases，随后进行高效液相色谱分级分离、部分氨基酸测序以及对标记肽段的质谱分析，结果显示两种HGPRTases各自的四条肽段被ox-GMP标记。这些研究得出的结论表明，人酶蛋白的两个区段，即Ser 4 - Arg 47和Ser 91 - Arg 100，以及血吸虫酶中的一个区域，即Gly 95 - Lys 133，被ox-GMP标记。由于人HGPRTase的ox-GMP标记可被GMP或磷酸核糖焦磷酸（PRibPP）有效阻断，而血吸虫HGPRTase的ox-GMP标记仅被GMP抑制[卡纳内，J.，克雷格，S. P.，三世，& 王，C. C.（1994年）《欧洲生物化学杂志》223卷，595 - 601页]，人酶中两个被标记的肽段可能参与与GMP和PRibPP的结合，而血吸虫酶中的一个肽段可能仅与GMP结合有关。我们还证实了之前的一项观察结果[基奥，D. T.，埃默森，B. T.，& 德泽西，J.（1991年）《生物化学与生物物理学报》1096卷，95 - 100页]，即碘乙酸对人HGPRTase中Cys 22的羧甲基化作用受到PRibPP的抑制。我们还证明，碘乙酸对血吸虫HGPRTase中Cys 25的羧甲基化作用被PRibPP特异性阻断。显然，两种酶的N端区域都参与PRibPP的结合。ox-GMP标记人酶的N端区域而不标记血吸虫酶，以及PRibPP可保护人酶免受ox-GMP标记而不能保护血吸虫酶，这两个事实都表明，人HGPRTase中氨基端的PRibPP结合位点可能与GMP结合位点紧密相邻，而在血吸虫HGPRTase中并非如此。人和血吸虫HGPRTases活性位点之间的这种明显差异可被进一步利用，为抗血吸虫化疗提供潜在机会。
