Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by 13C NMR and correlation with kinetic studies.
Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by 13C NMR and correlation with kinetic studies.
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葡萄球菌核酸酶活性位点氨基酸：13C NMR 测定酪氨酸和精氨酸的 pH 依赖性以及与动力学研究的相关性。
DOI：
10.1021/bi00431a023
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Markley,JL
中科院分区：
文献类型：
--
作者：
Grissom,CB;Markley,JL
1988年10月21日收到的修订稿件摘要：葡萄球菌核酸酶动力学参数的pH和温度依赖性(EC 3.1。4.7)，用三种含有3‘-取代基不同的DNA类似物的对-硝基苯基磷酸酯进行了检测。对于野生型(Foggi变异体)核酸酶(核酸酶wt)和底物胸苷S‘-磷酸5’-(/>；硝基苯磷酸)(PNPdTp)、胸腺嘧啶核苷3‘-甲基膦酸5’-(对硝基苯磷酸)(PNPdTp*Me)和胸苷5‘-(/t-硝基苯磷酸)(PNPdT)，kQIL在13min~(-1)时几乎保持不变。当酪氨酸-85被定点突变为苯丙氨酸(核酸酶Y85F)时，除PNPdTp下降到1min‘1外，fccat在约13min“1保持不变。对于核酸酶Y85F，PNPdTp和PNPdTp*me的kcal/Km分别为19.5和25 mm”1 min“1。以PNPdTp为底物，在0.3Mkc1和H20中的pKa值分别为8.94和9.67，呈现钟形的kcat/kmVS曲线。当酪氨酸-85改变为苯丙氨酸(核酸酶Y85F)或底物3‘-磷酸单酯改变为3’-甲基膦酸(PNPdTp*Me)时，在9.67处的PXA消失，而在10.1处出现新的PKA。这表明pKa为9.67的kQal/Km的拐点源于酪氨酸-85的电离，该电离氢键连接到具有该取代基的底物的二价3‘-磷酸单酯。在kcat/Km随pH的变化曲线中，两个去质子化步骤的电离热焓为5kcal/mol。用~(13)C核磁共振测定了精氨酸和酪氨酸残基的pKa值。蛋白质在所有碳原子上均以20%~(13)C均匀富集，在精氨酸残基的胍基碳上以90%~(13)C均匀富集。蛋白质中的五种精氨酸在H20和0.3Mkc1中的Ka值都大于11.6。这就消除了精氨酸作为碱性催化剂的可能性，这种催化剂可以使H20去质子化，从而促进对磷的亲核攻击。结果不排除精氨酸作为酸性催化剂的候选，该酸性催化剂在产品释放之前质子化5‘-核糖醇氧化物。通过比较核酸酶Wt和核酸酶Y85F的~(13)C核磁共振谱，对酪氨酸-85的酚羟基碳进行了归属。核酸酶Y85F的结构没有受到这种取代的显著影响。酪氨酸-85在H20和0.3Mkc1中的光谱观察pKa为9.53±0.05，与kcaJKm pH谱中碱性侧的pKa为9.67相似。这种pKa值与没有其他候选者之间的相关性表明，酪氨酸-85的电离是含有二价3‘-磷酸单酯的底物的kcal/kM与pH曲线中的pKa。这一结论与酪氨酸-85作为底物3‘-磷酸单酯氢键供体的作用是一致的。葡萄球菌核酸酶[核糖核酸(2’-脱氧核糖核酸盐)3‘-核苷酸水解酶，EC 3.1。催化S‘-磷酸与核糖环5’-氧之间的核糖基和2‘-脱氧核糖核苷酸的水解性切割。该酶已被深入研究，是阐明蛋白质结构和催化活性之间关系的原型(Hazen&Cotton，1978；Tucker等人，1979a-c)。详细了解电子邮件的催化作用
Revised Manuscript Received October 21, 1988 abstract: The pH and temperature dependence of the kinetic parameters of staphylococcal nuclease (EC 3.1. 4.7) have been examined with three p-nitrophenyl phosphate containing DNA analogues that vary as to 3'-substituent. With wild-type (Foggi variant) nuclease (nuclease wt) and the substrates thymidine S'-phosphate 5'-(/> nitrophenyl phosphate)(PNPdTp), thymidine 3'-methylphosphonate 5'-(p-nitrophenyl phosphate)(PNPdTp* Me), and thymidine 5'-(/t-nitrophenyl phosphate)(PNPdT), kQil remains nearly constant at 13 min" 1. However, k^ JK^ with nuclease wt varies considerably: 413, 13, and 0.52 mM" 1 min'1 with PNPdTp, PNPdTp* Me, and PNPdT, respectively. When tyrosine-85 is changed to phenylalanine (nuclease Y85F) by site-directed mutagenesis, fccat is unchanged at about 13 min" 1, except with PNPdTp where it drops to 1 min'1. With nuclease Y85F, kcal/Km is 19.5 and 25 mM" 1 min" 1 with PNPdTp and PNPdTp* Me, respectively. With PNPdTp as the substrate, a bell-shaped kcat/Km vs pH profile is seen with pKa values at 8.94 and 9.67 in 0.3 Mkc1 and H20. The pXa at 9.67 disappears, and a new pKa appears at 10.1 whentyrosine-85 is changed to phenylalanine (nuclease Y85F) or when the substrate 3'-phospho-monoester is changed to a 3'-methylphosphonate (PNPdTp* Me). This suggests that the inflection in kQal/Km with pKa at 9.67 arises from ionization of tyrosine-85, which hydrogen bonds to the divalent 3'-phospho-monoester of substrates with this substituent. The enthalpy of ionization of both deprotonation steps in the kcat/Km versus pH profile is 5 kcal/mol. 13C NMR has been used to determine the pKa values of the arginine and tyrosine residues. The protein was enriched uniformly with 20% 13C at all carbons and specifically with 90% 13C at the guanidino carbon of the arginine residues. All five arginines in the protein havepKa values greater than 11.6 in H20 and 0.3 Mkc1. This eliminates arginine as a candidate for the basic catalyst that deprotonates H20 to facilitate nucleophilic attack on phosphorus. The results do not rule out arginine as a candidate for the acidic catalyst that protonates the 5'-ribose alkoxide prior to product release. The phenolic hydroxyl carbon of tyrosine-85 has been assigned by comparing the 13C NMR spectrum of nuclease wt and nuclease Y85F. Thestructure of nuclease Y85F is not perturbed significantly by this substitution. Tyrosine-85 has a spectroscopically observed pKa of 9.53±0.05 in H20 and 0.3 Mkc1, which is similar to the basic-side pKa of 9.67 seen in the kcaJKm pH profile. This correlation between pKa values along with the absence of other candidates indicates that the ionization of tyrosine-85 is the pKa seen in the kcal/Km vs pH profile for substrates with a divalent 3'-phosphomonoester. This conclusion is consistent with the proposed role of tyrosine-85 as a hydrogen-bond donor to the 3'-phosphomonoester of substrates poised for exonucleolytic hydrolysis.Staphylococcal nuclease [ribonucleate (2'-deoxyribo-nucleate) 3'-nucleotidohydrolase, EC 3.1. 4.7] catalyzes the hydrolytic cleavage of ribo-and 2'-deoxyribonucleotides be-tween the S'-phosphate and the 5'-oxygen of the ribose ring. The enzyme has been studied thoroughly and is a prototype for elucidation of the relationship between protein structure and catalytic activity (Hazen & Cotton, 1978; Tucker et al., 1979a-c). A detailed understanding of the catalytic role of