Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase,Biochemistry

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Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase
Biochemistry ( IF 2.9 ) Pub Date : 2022-08-09 , DOI: 10.1021/acs.biochem.2c00298
Joshua H Hakala ₁ , Amanda J Laseke ₁ , Anya L Koza ₁ , Martin St Maurice ₁

Affiliation

Biotin-dependent enzymes employ a carrier domain to efficiently transport reaction intermediates between distant active sites. The translocation of this carrier domain is critical to the interpretation of kinetic and structural studies, but there have been few direct attempts to investigate the dynamic interplay between ligand binding and carrier domain positioning in biotin-dependent enzymes. Pyruvate carboxylase (PC) catalyzes the MgATP-dependent carboxylation of pyruvate where the biotinylated carrier domain must translocate ∼70 Å from the biotin carboxylase domain to the carboxyltransferase domain. Many prior studies have assumed that carrier domain movement is governed by ligand-induced conformational changes, but the mechanism underlying this movement has not been confirmed. Here, we have developed a system to directly observe PC carrier domain positioning in both the presence and absence of ligands, independent of catalytic turnover. We have incorporated a cross-linking trap that reports on the interdomain conformation of the carrier domain when it is positioned in proximity to a neighboring carboxyltransferase domain. Cross-linking was monitored by gel electrophoresis, inactivation kinetics, and intrinsic tryptophan fluorescence. We demonstrate that the carrier domain positioning equilibrium is sensitive to substrate analogues and the allosteric activator acetyl-CoA. Notably, saturating concentrations of biotin carboxylase ligands do not prevent carrier domain trapping proximal to the neighboring carboxyltransferase domain, demonstrating that carrier domain positioning is governed by conformational selection. This model of carrier domain translocation in PC can be applied to other multi-domain enzymes that employ large-scale domain motions to transfer intermediates during catalysis.

中文翻译：

构象选择控制金黄色葡萄球菌丙酮酸羧化酶中载体结构域的定位

生物素依赖性酶利用载体结构域在遥远的活性位点之间有效地运输反应中间体。该载体结构域的易位对于动力学和结构研究的解释至关重要，但很少有人直接尝试研究生物素依赖性酶中配体结合和载体结构域定位之间的动态相互作用。丙酮酸羧化酶 (PC) 催化丙酮酸的 MgATP 依赖性羧化，其中生物素化载体结构域必须将 ∼70 Å 从生物素羧化酶结构域易位至羧基转移酶结构域。许多先前的研究假设载体结构域的运动是由配体诱导的构象变化控制的，但这种运动的机制尚未得到证实。在这里，我们开发了一种系统，可以直接观察配体存在和不存在时 PC 载体结构域的定位，与催化周转无关。我们合并了一个交联陷阱，当载体结构域位于邻近的羧基转移酶结构域附近时，该交联陷阱报告载体结构域的结构域间构象。通过凝胶电泳、失活动力学和内在色氨酸荧光监测交联。我们证明载体结构域定位平衡对底物类似物和变构激活剂乙酰辅酶A敏感。值得注意的是，生物素羧化酶配体的饱和浓度并不能阻止载体结构域捕获邻近的羧基转移酶结构域，这表明载体结构域的定位受构象选择的控制。 PC 中的载体结构域易位模型可应用于其他多结构域酶，这些酶在催化过程中采用大规模结构域运动来转移中间体。

更新日期：2022-08-09

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