Identification of Amino Acid Residues Critical for Catalysis and Cosubstrate Binding in the Flavonol 3-Sulfotransferase (*)
Identification of Amino Acid Residues Critical for Catalysis and Cosubstrate Binding in the Flavonol 3-Sulfotransferase (*)
复制标题
鉴定对黄酮醇 3-磺基转移酶中的催化和共底物结合至关重要的氨基酸残基 (*)
DOI：
--
复制
发表时间：
1995
影响因子：
4.8
通讯作者：
L. Varin
中科院分区：
文献类型：
--
作者：
F. Marsolais;L. Varin
通过比较植物和动物硫转移酶（ST）的氨基酸序列，确定了4个保守性较好的区域，先前的实验证据表明，区域I和IV可能参与了共底物3 ' -磷酸腺苷5 ' -硫酸磷（PAPS）的结合。此外，IV区与富含甘氨酸的磷酸结合环（P-loop）基序同源，已知在几个蛋白家族中参与核苷酸磷酸结合。本研究通过植物黄酮醇3-ST的定点诱变研究了这两个区域内氨基酸残基的功能。在I区，我们的研究结果确定赖氨酸对催化作用至关重要，因为用丙氨酸替代该残基导致比活性降低300倍，而用精氨酸保守替代后观察到比活性降低15倍。用[yS]PAPS对K59R和K59A进行光亲和标记，发现共底物结合不需要赖氨酸。然而，K59A突变体对3 ' -磷酸腺苷5 ' -磷酸(PAP)-琼脂糖的亲和力降低，这表明Lys可能在反应过程中参与了中间体的稳定。在IV区，所有的Argy替换都导致比活性显著降低。Argy的保守和非保守替代导致与[yS]PAPS的弱光亲和标记，R276A/T73A和R276E酶对pap琼脂糖的亲和性降低，表明需要Argy侧链结合共底物。突变酶在残基Lys、Glyy和Lys处的动力学常数分析证实了IV区参与了共底物的结合。
The comparison of the deduced amino acid sequences of plant and animal sulfotransferases (ST) has allowed the identification of four well conserved regions, and previous experimental evidence suggested that regions I and IV might be involved in the binding of the cosubstrate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Moreover, region IV is homologous to the glycine-rich phosphate binding loop (P-loop) motif known to be involved in nucleotide phosphate binding in several protein families. In this study, the function of amino acid residues within these two regions was investigated by site-directed mutagenesis of the plant flavonol 3-ST. In region I, our results identify Lys as critical for catalysis, since replacement of this residue with alanine resulted in a 300-fold decrease in specific activity, while a 15-fold reduction was observed after the conservative replacement with arginine. Photoaffinity labeling of K59R and K59A with [yS]PAPS revealed that Lys is not required for cosubstrate binding. However, the K59A mutant had a reduced affinity for 3′-phosphoadenosine 5′-phosphate (PAP)-agarose, suggesting that Lys may participate in the stabilization of an intermediate during the reaction. In region IV, all substitutions of Argy resulted in a marked decrease in specific activity. Conservative and unconservative replacements of Argy resulted in weak photoaffinity labeling with [yS]PAPS and the R276A/T73A and R276E enzymes displayed reduced affinities for PAP-agarose, suggesting that the Argy side chain is required to bind the cosubstrate. The analysis of the kinetic constants of mutant enzymes at residues Lys, Glyy, and Lys allowed to confirm that region IV is involved in cosubstrate binding.
登录
查看更多内容
芳基磺基转移酶 IV 的亲和标记。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Zheng,Y;Bergold,A;Duffel,MW
通讯作者：
Duffel,MW
大鼠肝脏米诺地尔磺基转移酶 cDNA 的序列分析、体外翻译和表达。
影响因子：
3.6
作者：
Hirshey,SJ;Dooley,TP;Reardon,IM;Heinrikson,RL;Falany,CN
通讯作者：
Falany,CN
来自大鼠肝脏的胆汁酸磺基转移酶 I 硫酸化胆汁酸和 3-羟基类固醇：纯化、N 末端氨基酸序列和动力学特性。
影响因子：
6.5
作者：
Barnes,S;Buchina,ES;King,RJ;McBurnett,T;Taylor,KB
通讯作者：
Taylor,KB
影响因子：
4.1
作者：
COMER, KA;FALANY, JL;FALANY, CN
通讯作者：
FALANY, CN
3-磷酸腺苷-5-磷酸硫酸盐：磺基转移酶的光亲和配体。
影响因子：
3.6
作者：
Otterness,DM;Powers,SP;Miller,LJ;Weinshilboum,RM
通讯作者：
Weinshilboum,RM