On the specificity and stability of an enzyme that hydrolyzes N-substituted aminoacyl-transfer RNA's.

In an earlier investigation, we observed that Cbz-phe-tRNA' was rapidly hydrolyzed by a crude supernatant fraction from E. coli, whereas unsubstituted phe-tRNA was relatively stable under the same conditions.2Subsequent studies, reported here, have shown this hydrolytic enzyme (or enzymes) to have the following general specificity: it cleaves N-substituted amino acid residues from N-substituted AA-tRNA's but does not attack unsubstituted AA-tRNA.It thus appears to be the enzyme recently described by Cuzin et al.3We were led, independently, to an investigation of this enzyme by our studies of the initiation of protein synthesis in model systems, and we wish to report two relevant findings.First, there is a significant exception to the specificity as outlined above: F-met-tRNA, the normal initiator of protein synthesis in E. coli,4is not cleaved although it is an N-substituted AA-tRNA.Second, we have found the hydrolytic activity to be a highly concentrated contaminant of our preparations of initiation factors,5 both crude and purified, which were isolated by a widely employed procedure.6In view of its specificity, the enzyme seriously interferes with experiments on the initiation of protein synthesis in which other N-substituted AA-tRNA's are employed as models of F-met-tRNA.We have therefore developed a simple method of eliminating the activity from preparations of initiation factors.Cuzin et al.' have proposed that the enzyme may catalyze the terminal release of completed protein chains from tRNA.We wish to point out that its specificity is also compatible with an additional, corrective, function; this is discussed below.