Fast kinetic study of yeast phenylalanyl-tRNA synthetase: role of tRNAPhe in the discrimination between tyrosine and phenylalanine.
An extensive study of the discrimination between phenylalanine and tyrosine by yeast phenylalanyl-tRNA synthetase was carried out in the presence of native tRNAPhe. Our results clearly show that, besides the previously reported dissociation of tyrosyl adenylate from the enzyme template [Lin, S. X., Baltzinger, M., & Remy, P. (1983) Biochemistry 22, 681-689], two more correction processes are involved in the rejection of tyrosine in the presence of tRNAPhe. A minor part of the misactivated tyrosine is indeed transferred to tRNAPhe, but the resulting misaminoacylated tRNA is very rapidly hydrolyzed (kh approximately equal to 60 s-1), as it has already been shown for other systems. However, the major part of the misactivated tyrosine is rejected as the result of a pretransfer correction consisting of the fast hydrolysis (k'h approximately equal to 20 s-1) of the enzyme-bound noncognate adenylate induced by the binding of native tRNAPhe. The transfer step itself is found to be non-specific, as the rate constant is almost the same for phenylalanine and tyrosine. This result is supported by the observation that tyrosine and phenylalanine are also transferred at the same rate to tRNAPheox-red. It is shown that the integrity of the 3'-terminal adenosine of the tRNA is critical for triggering the pretransfer hydrolysis of enzyme-bound noncognate aminoacyl adenylate. A detailed kinetic analysis is presented that shows that the observed rate constant of tRNAPhe tyrosylation and the rate of disappearance of enzyme-tyrosyl adenylate complex are in fact apparent rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism*; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism*
D012346:RNA, Transfer, Amino Acyl; D014443:Tyrosine; D010649:Phenylalanine; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase