Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine.
Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine.
复制标题
腺苷酸激酶的机制。
DOI：
10.1021/bi00479a007
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Tsai,MD
中科院分区：
文献类型：
--
作者：
Yan,HG;Shi,ZT;Tsai,MD
俄亥俄州立大学化学系和俄亥俄州立大学生物化学项目，哥伦布，俄亥俄州 43210 收稿日期：1990 年 2 月 14 日；修订稿于 1990 年 4 月 4 日收到摘要：用赖氨酸或蛋氨酸替换腺苷酸激酶 (AK) 的精氨酸 138 导致 fccat 降低 104 倍，Km 增加 10-20 倍，并且解离常数变化相对较小。然后进行质子核磁共振（NMR）研究以获得结构信息以定量解释动力学数据。由于赖氨酸突变体 (R138K) 代表保守突变，对动力学影响惊人，因此结构研究集中在野生型 (WT) 和 R138K。结果和结论总结如下：(i)WT和R138K的芳香族自旋系统由全相关光谱法(TOCSY)指定。芳香质子化学位移、一维光谱、TOCSY 和核奥沃豪瑟增强光谱 (NOESY) 的比较表明，R138K 的构象相对于 WT 几乎没有受到干扰。因此，Arg-138 对于三级结构并不重要。(ii) 用 AMP 和 MgATP 进行的质子 NMR 滴定表明，WT 和 R138K 之间的底物结合亲和力和底物诱导的构象变化几乎相同。因此，精氨酸-138不应参与稳定二元复合物中的第一底物。(iii)在WT和R138K复合物与反应混合物的质子NMR谱之间观察到显着差异，这与R138K的Km值的扰动一致。使用“静态反应混合物”——P1，P5-双（5/-腺苷）五磷酸盐（MgAP5A）详细分析了差异。 WT+ MgAP5A 和 R138K+ MgAP5A 的芳香族自旋系统是根据各种二维光谱部分分配的。结果表明，WT和R138K的三元复合物之间的构象差异很小。(iv)脂肪族侧链和芳香族质子之间的NOESY交叉峰的定性比较表明游离WT和游离R138K之间的模式几乎相同。对于 WT+ MgAP5A 和 R138K+ MgAP5A，模式也非常相似，但可以观察到一些小的变化。这些观察结果再次证实了芳香区定量分析得出的结论，(v)上述动力学和结构结果得出的结论是，Arg-138使三元配合物稳定1.4—1.8 kcal/mol，并使过渡态稳定至少7 kcal/mol，并且Arg-138的关键功能作用不能被赖氨酸取代，(vi)由于Arg-138与先前提出的底物位点相距较远NMR 研究 [Mildvan, A. S., & Fry, D. C.(1987) Adv.埃里济莫尔。相关。领域分子。生物。 58, 241-313]，该模型需要认真修改，(vii) 结合的 MgAP5A 的茶腺嘌呤和 Hr 质子是从 WT 和突变体的 orie 维和 NOESY 光谱中明确指定的，这表明组氨酸 36 和结合的 MgAP5A 共振之间没有可检测到的 NOE，这与最近关于猪肌肉 AK 的报告相反 [Rósch, P., Klaus, W., Auer, M., & Goody, R. S.(199) Biochemistry 28, 4318-4325]，尽管定点诱变现已成为鉴定催化重要酶残基的常用方法，但它经常产生新的争议而不是解决问题。就腺苷酸激酶 (AK) 而言，根据…的动力学和有限结构数据，现在建议 1 个三个残基与 ATP 的磷酰基相互作用。
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210 Received February 14, 1990; Revised Manuscript Received April 4, 1990 abstract: Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in fccat by a factor of 104, increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutationwith surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows:(i) The aromatic spin systems Of WT and R138K were assigned from total correlatedspectroscopy (TOCSY). Comparison of the chemical shifts of aromatic protons, one-dimensional spectra, TOCSY, and nuclear Overhauser enhanced spectroscopy (NOESY) indicated that the conformation of R138K was almost unperturbed relative to that of WT. Thus Arg-138 is not important for the tertiary structure,(ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-inducedconformational changes are nearly identical between WT and R138K. Thus arginine-138 should not be involved in stabilizing the first substrate in the binary complex,(iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reactionmixture, which agrees with the perturbation in the Km values of R138K. The differences were analyzed in detail by using a “static reaction mixture”—P1, P5-bis (5/-adenosyl) pentaphosphate (MgAP5A). The aromatic spin systems of WT+ MgAP5A and R138K+ MgAP5A were partially assigned from various two-dimensional spectra. The results suggest that the conformational differencesbetween the ternary complexes of WT and Rl38K are only minor,(iv) Qualitative comparison of the NOESY cross peaks between aliphatic side chains and aromatic protons indicates that the patterns are almost identical between free WT and free R138K. For WT+ MgAP5A and R138K+ MgAP5A the patterns are also very similar, but some small shifts can be observed. Theseobservations reaffirm the conclusions drawn from the quantitative analysis of the aromatic region,(v) The above kinetic and structural results led to the conclusion that Arg-138stabilizes the ternary complexes by 1.4—1.8 kcal/mol and stabilizes the transition state by at least 7 kcal/mol and that the critical functional role of Arg-138 cannot be replaced by lysine,(vi) Since Arg-138 is distant from the substrate sites proposed from previous NMR studies [Mildvan, A. S., & Fry, D. C.(1987) Adv. Erizymol. Relat. Areas Mol. Biol. 58, 241-313], serious revision will be required for this model,(vii) Theadenine and Hr protons of bound MgAP5A were unequivocally assigned from the orie-dimensional and NOESY spectra of WT and mutants, which indicated no detectable NOE between histidine-36 and bound MgAP5A resonances as opposed to a recent report on porcine muscle AK [Rósch, P., Klaus, W., Auer, M., & Goody, R. S.(199) Biochemistry 28, 4318-4325], Although site-directed mutagenesis has now become a common approach in identifying catalytically important res-idues of enzymes, it has often generated new controversies rather than solved problems. In the case of adenylate kinase (AK), 1 three residues have now beensuggested to interact with the-phosphoryl group of ATP on the basis of the kinetic and limited structural data of …
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化学修饰和定点诱变证明了精氨酸 88 在大肠杆菌腺苷酸激酶中的结构和催化作用。
影响因子：
4.8
作者：
Jochen Reinstein;Anne;Thierry Rose;Alfred Wittinghofer;Isabelle Saint−Girons;Octavian Bârzu;W. Surewicz;Henry H. Mantsch
通讯作者：
Henry H. Mantsch
DOI：
10.1111/j.1432-1033.1986.tb10132.x
发表时间：
1986-11
期刊：
European journal of biochemistry
影响因子：
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作者：
G. Schulz;E. Schiltz;A. Tomasselli;R. Frank;M. Brune;A. Wittinghofer;R. Schirmer
通讯作者：
G. Schulz;E. Schiltz;A. Tomasselli;R. Frank;M. Brune;A. Wittinghofer;R. Schirmer
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影响因子：
2.9
作者：
W. Klaus;M. Scharf;S. Zimmermann;P. Rösch
通讯作者：
P. Rösch
ATP 酶和磷酸激酶测定中的腺苷酸激酶合成抑制剂。
DOI：
--
发表时间：
1975
期刊：
European Journal of Biochemistry
影响因子：
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作者：
P. Feldhau;T. Fröhlich;R. Goody;M. Isakov;R. Schirmer
通讯作者：
R. Schirmer
P1-(lin-苯并-5-腺苷)-P4-(5-腺苷)四磷酸和P1-(lin-苯并-5-腺苷)-P5-(5-腺苷五磷酸)抑制腺苷酸激酶。
影响因子：
2.9
作者：
P. Vanderlijn;J. Barrio;N. Leonard
通讯作者：
N. Leonard