Enzymatic Hydrolysis of N-Substituted Aminoacyl Transfer Ribonucleic Acid in Yeast

An N-substituted aminoacyl transfer RNA hydrolase (ribonuclease free) has been isolated, purified 230-fold, and crystallized from yeast supernatant. The purified enzyme is activated by 10-3 and 10-2 m MgCl2 and strongly inhibited by 10-1 m MgCl2. The hydrolase is also strongly inhibited by 10-2 m ethylenediaminetetraacetic acid, and the inhibition can be completely overcome with 10-2 m MgCl2. The yeast hydrolase is competitively inhibited by uncharged yeast tRNA. Purified yeast ribosomal RNA does not inhibit the enzyme. The hydrolase does not show a strict species specificity since the tRNA of both yeast and Escherichia coli can be used to form substrate. N-Acetyl-valyl-adenosine is not a substrate for the hydrolase and N-acetyl-valyl-oligonucleotide is hydrolyzed very slowly. Although the free aminoacyl-tRNAs tested are resistant or very slowly hydrolyzed, the derivatives N-acetylaminoacyl-tRNA, N-benzoyl-glycyl-glycyl-phenylalanyl-tRNA, and N-benzoyl-glycyl-glycyl-glycyl-phenylalanyl-tRNA are all readily hydrolyzed by the purified enzyme. Yeast oligolysyl-tRNA is very slowly hydrolyzed by the enzyme. N-Substituted amino acids and tRNAs are the products of hydrolysis of N-substituted aminoacyl-tRNAs. Enzymatically deacylated tRNAs can be recharged with their corresponding amino acids.