Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

Guanosine 5′-monophosphate (GMP) synthetases,
enzymes that
catalyze the conversion of xanthosine 5′-monophosphate (XMP)
to GMP, are composed of two different catalytic units, which are either
two domains of a polypeptide chain or two subunits that associate
to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes
glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase)
unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound
ATPPase allosterically activates GATase, and the ammonia thus generated
is tunneled to the ATPPase active site where it reacts with AMP-XMP
generating GMP. In ammonia channeling enzymes reported thus far, a
tight complex of the two subunits is observed, while the interaction
of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism
of allostery and substrate channeling largely unclear. Here, we present
a mechanistic model encompassing the various steps in the catalytic
cycle of MjGMPS based on biochemical experiments, crystal structure,
and cross-linking mass spectrometry guided integrative modeling. pH
dependence of enzyme kinetics establishes that ammonia is tunneled
across the subunits with the lifetime of the complex being ≤0.5
s. The crystal structure of the XMP-bound ATPPase subunit reported
herein highlights the role of conformationally dynamic loops in enabling
catalysis. The structure of MjGMPS derived using restraints obtained
from cross-linking mass spectrometry has enabled the visualization
of subunit interactions that enable allostery under catalytic conditions.
We integrate the results and propose a functional mechanism for MjGMPS
detailing the various steps involved in catalysis.

鸟苷酸5'-单磷酸酯（GMP）合成酶，一类催化黄嘌呤5'-单磷酸酯（XMP）转化为GMP的酶，由两个不同的催化单位组成，这两个单位可以是多肽链的两个结构域，或者两个相互结合形成复合体的亚基。谷氨酰胺转氨酶（GATase）单位通过水解谷氨酰胺产生氨，而三磷酸腺苷焦磷酸酶（ATPPase）单位催化AMP-XMP中间体的形成。结合底物的ATPPase通过变构激活GATase，从而产生的氨通过隧道效应传递至ATPPase的活性位点，与AMP-XMP反应生成GMP。在迄今为止报道的氨通道酶中，两个亚基形成了一个紧密的复合体，而甲烷古菌甲烷热球菌（Methanocaldococcus jannaschii）GMP合成酶（MjGMPS）的两个亚基之间的相互作用是瞬时的，其变构和底物通道的潜在机制尚不明确。在此，我们基于生化实验、晶体结构和交联质谱引导的整合建模，提出一个包含MjGMPS催化循环中各个步骤的机制模型。酶动力学对pH的依赖性表明，氨通过亚基隧道效应传递，复合体的寿命≤0.5秒。本文报道的XMP结合的ATPPase亚基的晶体结构突出了构象动态环在催化中的作用。利用来自交联质谱的约束得到的MjGMPS结构，实现了在催化条件下亚基相互作用的可视化。我们将结果进行整合，并提出了MjGMPS的功能机制，详细阐述了催化过程中涉及的各个步骤。