Pitera J W, Munagala N R, Wang C C, Kollman P A
Graduate Group in Biophysics and Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, California 94143-0446, USA.
Biochemistry. 1999 Aug 10;38(32):10298-306. doi: 10.1021/bi990250w.
We present molecular dynamics (MD) simulations on two enzymes: a human hypoxanthine-guanine-phosphoribosyltransferase (HGPRTase) and its analogue in the protozoan parasite Tritrichomonas foetus. The parasite enzyme has an additional ability to process xanthine as a substrate, making it a hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) [Chin, M. S., and Wang, C. C. (1994) Mol. Biochem. Parasitol. 63 (2), 221-229 (1)]. X-ray crystal structures of both enzymes complexed to guanine monoribosyl phosphate (GMP) have been solved, and show only subtle differences in the two active sites [Eads et al. (1994) Cell 78 (2), 325-334 (2); Somoza et al. (1996) Biochemistry 35 (22), 7032-7040 (3)]. Most of the direct contacts with the base region of the substrate are made by the protein backbone, complicating the identification of residues significantly associated with xanthine recognition. Our calculations suggest that the broader specificity of the parasite enzyme is due to a significantly more flexible base-binding region, and rationalize the effect of two mutations, R155E and D163N, that alter substrate specificity [Munagala, N. R., and Wang, C. C. (1998) Biochemistry 37 (47), 16612-16619 (4)]. In addition, our simulations suggested a double mutant (D106E/D163N) that might rescue the D163N mutant. This double mutant was expressed and assayed, and its catalytic activity was confirmed. Our molecular dynamics trajectories were also used with a structure-based design program, Pictorial Representation Of Free Energy Changes (PROFEC), to suggest parasite-selective derivatives of GMP. Our calculations here successfully rationalize the parasite-selectivity of two novel inhibitors derived from the computer-aided design of Somoza et al. (5) and demonstrate the utility of PROFEC in the design of species-selective inhibitors.
我们展示了对两种酶的分子动力学（MD）模拟：一种是人类次黄嘌呤 - 鸟嘌呤 - 磷酸核糖转移酶（HGPRTase），另一种是原生动物寄生虫胎儿三毛滴虫中的类似物。该寄生虫酶具有额外将黄嘌呤作为底物进行加工的能力，使其成为次黄嘌呤 - 鸟嘌呤 - 黄嘌呤磷酸核糖转移酶（HGXPRTase）[Chin, M. S., and Wang, C. C. (1994) Mol. Biochem. Parasitol. 63 (2), 221 - 229 (1)]。已解析了两种酶与鸟嘌呤单磷酸核糖（GMP）复合的X射线晶体结构，并且显示两个活性位点仅有细微差异[Eads等人(1994) Cell 78 (2), 325 - 334 (2); Somoza等人(1996) Biochemistry 35 (22), 7032 - 7040 (3)]。与底物碱基区域的大多数直接接触是由蛋白质主链进行的，这使得鉴定与黄嘌呤识别显著相关的残基变得复杂。我们的计算表明，寄生虫酶更广泛的特异性归因于碱基结合区域显著更高的灵活性，并解释了改变底物特异性的两个突变R155E和D163N的作用[Munagala, N. R., and Wang, C. C. (1998) Biochemistry 37 (47), 16612 - 16619 (4)]。此外，我们的模拟提出了一个可能挽救D163N突变体的双突变体（D106E/D163N）。该双突变体被表达并进行了测定，其催化活性得到了证实。我们的分子动力学轨迹还与基于结构的设计程序“自由能变化的图形表示”（PROFEC）一起使用，以提出GMP的寄生虫选择性衍生物。我们在此的计算成功地解释了源自Somoza等人计算机辅助设计的两种新型抑制剂的寄生虫选择性(5)，并证明了PROFEC在物种选择性抑制剂设计中的实用性。
