Abstract
N(α)-acetylation is a common protein modification catalyzed by different N-terminal acetyltransferases (NATs). Their essential role in the biogenesis and degradation of proteins is becoming increasingly evident. The NAT hNaa50p preferentially modifies peptides starting with methionine followed by a hydrophobic amino acid. hNaa50p also possesses N(ε)-autoacetylation activity. So far, no eukaryotic NAT has been mechanistically investigated. In this study, we used NMR spectroscopy, bisubstrate kinetic assays, and product inhibition experiments to demonstrate that hNaa50p utilizes an ordered Bi Bi reaction of the Theorell-Chance type. The NMR results, both the substrate binding study and the dynamic data, further indicate that the binding of acetyl-CoA induces a conformational change that is required for the peptide to bind to the active site. In support of an ordered Bi Bi reaction mechanism, addition of peptide in the absence of acetyl-CoA did not alter the structure of the protein. This model is further strengthened by the NMR results using a catalytically inactive hNaa50p mutant.
Highlights
N␣-Acetylation is catalyzed by N-terminal acetyltransferases (NATs)
1C).This result is consistent with a model in which acetylated 1MLGP–RRR24 does not form a stable interaction with the hNaa50p-CoA complex, concordant with the lack of product inhibition observed in the kinetic experiments
We have presented enzyme kinetic and NMR data that point to an ordered ternary complex mechanism for the human Naa50p NAT activity
Summary
N␣-Acetylation is catalyzed by N-terminal acetyltransferases (NATs). The reaction mechanisms of NATs are unknown. hNaa50p is a member of the human NAT family. Significance: Bisubstrate inhibitors, mimicking a ternary complex, should function as specific inhibitors of human NATs. N␣-Acetylation is a common protein modification catalyzed by different N-terminal acetyltransferases (NATs). N␣-Acetylation is a common protein modification catalyzed by different N-terminal acetyltransferases (NATs) Their essential role in the biogenesis and degradation of proteins is becoming increasingly evident. We used NMR spectroscopy, bisubstrate kinetic assays, and product inhibition experiments to demonstrate that hNaa50p utilizes an ordered Bi Bi reaction of the Theorell-Chance type. In support of an ordered Bi Bi reaction mechanism, addition of peptide in the absence of acetyl-CoA did not alter the structure of the protein This model is further strengthened by the NMR results using a catalytically inactive hNaa50p mutant. Bisubstrate inhibitors, mimicking a ternary complex molecule, should function as highly specific and efficient inhibitors of hNaa50p and possibly other human NATs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
