posted on 2022-08-30, 13:36authored bySantosh Shivakumaraswamy, Sanjeev Kumar, Asutosh Bellur, Satya Dev Polisetty, Hemalatha Balaram
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.