HPLC-based quantification of in vitro N-terminal acetylation.

Protein N-terminal acetylation is a widespread modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs). The various NATs and their specific substrate specificities and catalytic mechanisms are far from fully understood. We here describe an in vitro method based on reverse-phase HPLC to quantitatively measure in vitro acetylation of NAT oligopeptide substrates, enabling the determination of NAT specificity as well as kinetic parameters.

Human protein N-terminal acetyltransferase hNaa50p (hNAT5/hSAN) follows ordered sequential catalytic mechanism: combined kinetic and NMR study.

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.