Rajamohan F, Pugmire M J, Kurinov I V, Uckun F M
Biotherapy Program, Parker Hughes Institute, St. Paul, Minnesota 55113, USA.
J Biol Chem. 2000 Feb 4;275(5):3382-90. doi: 10.1074/jbc.275.5.3382.
The Phytolacca americana-derived naturally occurring ribosome inhibitory protein pokeweed antiviral protein (PAP) is an N-glycosidase that catalytically removes a specific adenine residue from the stem loop of ribosomal RNA. We have employed molecular modeling studies using a novel model of PAP-RNA complexes and site-directed mutagenesis combined with bioassays to evaluate the importance of the residues at the catalytic site and a putative RNA binding active center cleft between the catalytic site and C-terminal domain for the enzymatic deadenylation of ribosomal RNA by PAP. As anticipated, alanine substitutions by site-directed mutagenesis of the PAP active site residues Tyr(72), Tyr(123), Glu(176), and Arg(179) that directly participate in the catalytic deadenylation of RNA resulted in greater than 3 logs of loss in depurinating and ribosome inhibitory activity. Similarly, alanine substitution of the conserved active site residue Trp(208), which results in the loss of stabilizing hydrophobic interactions with the ribose as well as a hydrogen bond to the phosphate backbone of the RNA substrate, caused greater than 3 logs of loss in enzymatic activity. By comparison, alanine substitutions of residues (28)KD(29), (80)FE(81), (111)SR(112), (166)FL(167) that are distant from the active site did not significantly reduce the enzymatic activity of PAP. Our modeling studies predicted that the residues of the active center cleft could via electrostatic interactions contribute to both the correct orientation and stable binding of the substrate RNA molecule in the active site pocket. Notably, alanine substitutions of the highly conserved, charged, and polar residues of the active site cleft including (48)KY(49), (67)RR(68), (69)NN(70), and (90)FND(92) substantially reduced the depurinating and ribosome inhibitory activity of PAP. These results provide unprecedented evidence that besides the active site residues of PAP, the conserved, charged, and polar side chains located at its active center cleft also play a critical role in the PAP-mediated depurination of ribosomal RNA.
美洲商陆来源的天然核糖体抑制蛋白商陆抗病毒蛋白（PAP）是一种N - 糖苷酶，它能催化从核糖体RNA的茎环中去除一个特定的腺嘌呤残基。我们采用分子建模研究，使用一种新型的PAP - RNA复合物模型，并结合定点诱变和生物测定法，来评估催化位点以及催化位点与C末端结构域之间假定的RNA结合活性中心裂隙处的残基，对于PAP催化核糖体RNA去嘌呤化的重要性。正如预期的那样，通过定点诱变将直接参与RNA催化去嘌呤化的PAP活性位点残基Tyr(72)、Tyr(123)、Glu(176)和Arg(179)替换为丙氨酸，导致脱嘌呤和核糖体抑制活性损失超过3个对数级。同样，保守活性位点残基Trp(208)被丙氨酸替换，这导致与核糖的稳定疏水相互作用丧失以及与RNA底物磷酸骨架的氢键丧失，使得酶活性损失超过3个对数级。相比之下，远离活性位点的残基(28)KD(29)、(80)FE(81)、(111)SR(112)、(166)FL(167)被丙氨酸替换并没有显著降低PAP的酶活性。我们的建模研究预测，活性中心裂隙处的残基可以通过静电相互作用，有助于底物RNA分子在活性位点口袋中正确定向和稳定结合。值得注意的是，活性中心裂隙处高度保守、带电荷和极性的残基，包括(48)KY(49)、(67)RR(68)、(69)NN(70)和(90)FND(92)被丙氨酸替换，大幅降低了PAP的脱嘌呤和核糖体抑制活性。这些结果提供了前所未有的证据，表明除了PAP的活性位点残基外，位于其活性中心裂隙处的保守、带电荷和极性侧链在PAP介导的核糖体RNA去嘌呤化中也起着关键作用。
