Kinetic proofreading is a reaction scheme with a structure more complicated than that of Michaelis kinetics, which leads to a proofreading for errors in the recognition of a correct substrate by an enzyme. We have measured the stoichiometry between ATP hydrolysis and tRNAIle charging, using the enzyme isoleucyl-tRNA synthetase [L-iso-leucine:tRNAIe ligase (AMP-forming), EC 6.1.1.5] and the amino acids isoleucine (correct) and valine (incorrect). The enzymatic deacylation of charged tRNA, which would normally prevent meaningful stoichiometry studies, was eliminated by the use of transfer factor Tu'GTP, (which binds strongly to charged tRNA) in the reaction mixture. For isoleucine, 1.5 ATP molecules are hydrolyzed per tRNA charged, but for valine, 270. These stoichiometry ratios are fundamental to kinetic proofreading, for the energy coupling is essential and proofreading is obtained only by departing from 1:1 stoichiometry between energy coupling and product formation. Within the known reaction pathway, these ratios demonstrate that kinetic proofreading induces a reduction in errors by a factor of 1/180. An overall error rate of about 10-4 for tRNA charging is obtained by a kinetic proofreading using a fundamental discrimination level of about 10-2, and is compatible with the low in vivo error rate of protein synthesis.Many biochemical reactions, particularly those associated with protein synthesis or DNA replication, exhibit high specificity in the selection between similar substrates. The overall error rate in selecting between two similar amino acids (1) in protein synthesis is believed to be about 3 in 104. The normal error rate in DNA synthesis (without post-replication repair) is about 1 in 108 or 109. These low error rates are biologically essential.The elementary description of specificity in biochemical reactions is based on discrimination in a Michaelis complex. [2]When C and D are sufficiently similar, AG will not be large, and fo may be smaller than would be biologically optimal. "Kinetic proofreading" (2) is a method of using twice (or more) the same Michaelis kinetic ability to distinguish between C and D, resulting in an error rate as small as fo2 (or higher power) instead of fo for a given AG. The essential features of kinetic proofreading are contained in the reaction schemeThe first reaction step reversibly forms the usual Michaelis complex. The second step is enzymatically coupled to an energy source, typically the hydrolysis of a nucleoside triphosphate, and is strongly enough driven to be essentially irreversible. (Cc)* is a high energy intermediate, which can decompose in two ways: to free enzyme plus product or to free enzyme and nonproduct C'. C' can be either the original substrate C or a chemically modified form thereof.This scheme is capable of proofreading. For equal concentrations of C and D, the discrimination against D in the initial Michaelis complex and in the formation of (Dc)* will be fo. If the kinetic constants are appropriate (and they can be made so without enhancing...
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