Inhibition of the puromycin reaction with benzamidine and related compounds

Abstract

The similarity between the mode of action of proteolytic enzymes and ribosomal peptidyltransferasecatalyzed peptide bond formation is of considerable current interest [1,2]. Peptidyltransferase can function essentially in three possible ways: (i) Via a double displacement mechanism involving an acyl-enzyme (ribosome) intermediate as found in serine proteases [3 ]; (ii) By a direct transfer of peptidyl residue to aminoacyl function without forming a covalent ribosomal intermediate or catalytic participation of the ribosome [4]; or ('tii) Via a concerted cyclic mechanism which includes both transfer of peptidyl moiety and proton [5] from a suitably oriented functional group on the ribosome. One approach to an evaluation of possibility (i) is to study a response of some known inhibitors of proteolytic enzymes in a suitable model system of peptidyltransferase-catalyzed peptide bond formation. Thus, it was reported [6] that phenylboronic acids, a group of chymotrypsin irthibitors [7], inhibited the fragment reaction and also, to a lesser extent, the reaction of N-Ac-Phe-tRNA with puromycin. Unlike proteases, which are fairly specific for distinct amino acids, ribosomal peptidyltransferase is capable of catalyzing peptide-bond formation with aminoacyl tRNAs derived from all 20 amino acids. It is then likely that peptidyltransferase can incorporate the active site features of proteolytic enzymes other than simply those of chymotrypsin. Therefore, it was of interest to study a group of aromatic amidines, which are known to inhibit trypsin [8] as possible inhibitors of peptidyltransferase. All amidine derivatives, L-arginine and sodium benzoate, used here, were commercial products of the highest available purity./3-Phenylethylboronic acid was prepared as in [9,10]. The pH of all inhibitor solutions was adjusted to 7.4 with HC1 or NaOH prior to the assay. Inhibition of the puromycin reaction was performed as in [11 ]. A typical reaction mixture conrained in 0.1 ml: 0.05 M Tris-HC1 (pH 7.4), 0.1 M NH4CI, 0.01 M MgC12, 4.0 A26o units of NH4-washed 70 S ribosomes from E. coli MRE 600 cells, 10 g poly(U), 0.20 A26o units of N-Ac-[14C]Phe-tRNA (~5200 cpm, spec. act. 0.84 nmol [~4C]phenylalanine/mg tRNA), puromycin 1 X 10 -s M and inhibitor at desired concentrations. Following the incubation at 37°C for 30 mill, the reaction was stopped by addition of 0.1 ml 0.1 M Be(NOa)2 in 0.3 M acetate buffer (pH 5.5) saturated with MgSO4 and the products were extracted with 1.5 ml ethyl acetate. Ethyl acetate phase (1 ml) was transferred to a scintillation vial and the radioactivity was determined in 10 ml Scinti Verse TM scintillation mixture (Fisher Scientific Co., Fair Lawn N J) in a Packard Tri-Carb liquid scintillation spectrometer at 73% counting efficiency. Percent inhibition represents the difference in ethyl acetate extracted N-acetyl-L[ ~4 C] phenylalanylpuromycin counts in the absence and in the presence of inhibitors. Further experimental details are provided in the figure legends.