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首页> 外文期刊>ACS Omega >Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides
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Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides

机译：残留网络参与腺嘌呤核苷酸对胱硫醚β-合酶结构域焦磷酸酶的变构调节

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摘要

Inorganic pyrophosphatase containing regulatory cystathionine β-synthase (CBS) domains (CBS-PPase) is inhibited by adenosine monophosphate (AMP) and adenosine diphosphate and activated by adenosine triphosphate (ATP) and diadenosine polyphosphates; mononucleotide binding to CBS domains and substrate binding to catalytic domains are characterized by positive cooperativity. This behavior implies three pathways for regulatory signal transduction — between regulatory and active sites, between two active sites, and between two regulatory sites. Bioinformatics analysis pinpointed six charged or polar amino acid residues of Desulfitobacterium hafniense CBS-PPase as potentially important for enzyme regulation. Twelve mutant enzyme forms were produced, and their kinetics of pyrophosphate hydrolysis was measured in wide concentration ranges of the substrate and various adenine nucleotides. The parameters derived from this analysis included catalytic activity, Michaelis constants for two active sites, AMP-, ATP-, and diadenosine tetraphosphate-binding constants for two regulatory sites, and the degree of activation/inhibition for each nucleotide. Replacements of arginine 295 and asparagine 312 by alanine converted ATP from an activator to an inhibitor and markedly affected practically all the above parameters, indicating involvement of these residues in all the three regulatory signaling pathways. Replacements of asparagine 312 and arginine 334 abolished or reversed kinetic cooperativity in the absence of nucleotides but conferred it in the presence of diadenosine tetraphosphate, without effects on nucleotide-binding parameters. Modeling and molecular dynamics simulations revealed destabilization of the subunit interface as a result of asparagine 312 and arginine 334 replacements by alanine, explaining abolishment of kinetic cooperativity. These findings identify residues 295, 312, and 334 as crucial for CBS-PPase regulation via CBS domains.
机译：含有调节性胱硫醚β-合酶（CBS）结构域（CBS-PPase）的无机焦磷酸酶被单磷酸腺苷（AMP）和二磷酸腺苷抑制，并被三磷酸腺苷（ATP）和多磷酸腺苷磷酸激活。与CBS结构域结合的单核苷酸和与催化结构域结合的底物的特征是正协同性。此行为暗示了调控信号传导的三种途径-调控位点与活性位点之间，两个活性位点之间以及两个调控位点之间。生物信息学分析指出，Hafniense CBS-PPase的六个带电荷或极性氨基酸残基可能对酶的调控很重要。产生了十二种突变酶形式，并在较宽的底物和各种腺嘌呤核苷酸浓度范围内测量了焦磷酸水解的动力学。从该分析中得出的参数包括催化活性，两个活性位点的Michaelis常数，两个调节位点的AMP-，ATP-和二磷酸腺苷四磷酸结合常数，以及每个核苷酸的激活/抑制程度。丙氨酸替代精氨酸295和天冬酰胺312将ATP从激活剂转化为抑制剂，并且实际上显着影响了上述所有参数，表明这些残基参与了所有三个调节信号通路。天冬酰胺312和精氨酸334的替换在没有核苷酸的情况下废除了或逆转了动力学的协同作用，但在四磷酸二腺苷的存在下赋予了它，而对核苷酸结合参数没有影响。建模和分子动力学模拟显示，由于天冬酰胺312和精氨酸334被丙氨酸替代，导致亚基界面不稳定，这说明动力学协同性的取消。这些发现确定了残基295、312和334对于通过CBS结构域调节CBS-PPase至关重要。

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