Two Glu/Asp residues cooperatively mediate an early step of ATP hydrolysis in GHKL ATPases MutL and GyrB
GHKL ATPases share a unique Bergerat ATP-binding fold and regulate diverse biological processes through ATP-dependent conformational changes. An early step of ATP hydrolysis in this family has been attributed to a single highly conserved glutamate residue proposed to function as the general base. However, mutations of this residue impair both the ATPase activity and ATP binding, complicating…
Glu/Asp residues work together to enable the initial stage of ATP breakdown in GHKL ATPases MutL and GyrB. These enzymes, which belong to a family that regulates various biological processes via conformational changes, share a distinctive Bergerat ATP-binding fold. Researchers found that a single glutamate residue, crucially conserved across these enzymes, was thought to act as a general base in ATP hydrolysis.
However, altering this residue affected both ATPase activity and ATP binding, making it challenging to determine its exact role. A closer look at high-resolution crystal structures of Aquifex aeolicus MutL and GyrB revealed a second acidic residue near a nucleophilic water molecule, where hydrogen bonding occurs. By conducting mutagenesis, ATPase and ATP-binding assays, and X-ray crystallography on these enzymes, scientists determined that maintaining the water's hydrogen bonding capability with the glutamate is sufficient for efficient ATP hydrolysis.
However, the proton-accepting capacity of at least one of the two acidic residues is necessary for optimal catalysis. This suggests that the glutamate primarily positions the nucleophilic water, while the activation of this water for catalysis involves a cooperative general base function provided by the two acidic residues. The researchers applied this understanding to human MutL homologs, PMS2 and MLH1, discovering that certain clinically observed variants have reduced ATPase activity, indicating functional impairment.
In summary, the study elucidates the catalytic mechanism of GHKL ATPases, offering a structural and functional framework for understanding disease-associated variants in these enzymes. Additionally, phylogenetic and ancestral state analysis suggest that the second acidic residue was likely present in the common ancestor of major GHKL ATPase lineages but was subsequently modified in a particular branch, including Hsp90, pointing to evolutionary adaptations in the catalytic mechanism.
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