Identifying the substrates and generating bisubstrate inhibitors of Gcn5-related N-acetyltransferases

Abstract

The Gcn5-related N-acetyltransferase (GNAT) superfamily of enzymes, which includes over 10,000 enzymes from all kingdoms of life, catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to a wide range of amine-containing substrates. The substrates for the vast majority of these enzymes are unknown.;We have demonstrated that chloroacetyl-CoA is a substrate for pro- and eukaryotic GNAT superfamily members that act on protein substrates, including the bacterial RimL protein, whose substrate is the ribosomal protein L12, and the yeast Hat1 histone acetyltransferase, whose substrate is histone H4. The chloroacetylated protein products of these reactions can be identified by their facile reaction with exogenously added thiol-containing nucleophiles. The thiol-containing compounds can be equipped with affinity and/or fluorescent tags that allow for the purification and identification of the chloroacetylated product. When the nucleophile added is CoA, the corresponding acetylCoAylated bisubstrate analog is generated. Incorporation of a his-tag on the acetyltransferase allows for the isolation of both the acetyltransferase and the tightly bound bisubstrate by nickel affinity chromatography. The above methods provided sufficient sensitivity and specificity to identify GNAT substrates in crude cell extracts.;Ribosomal protein S18 acetyltransferase (RimI), aminoglycoside acetyltransferase AAC(3)-IV, and spermine acetyltransferase can also utilize ClAcCoA as a substrate. The corresponding bisubstrate inhibitors, AcCoAylated S18 peptide, AcCoAylated tobramycin, and AcCoAylated spermine, were synthesized and used in co-crystallization and inhibition studies. Matthew Vetting from this laboratory solved the crystal structure of RimI with AcCoAylated S18 peptide. We also present a theoretical analysis of bisubstrate inhibition that allows the differentiation of random and ordered mechanism. Applying this analysis, AAC(3)-IV and RimI were found to have a random and ordered kinetic mechanisms, respectively.;The methods outlined above should allow one to identify substrate(s) for GNAT superfamily members that use acetylCoA, and the corresponding bisubstrate inhibitors can subsequently be synthesized for use in crystallization and kinetic studies.