Isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600

For discrimination between isoleucine and 19 other amino acids by isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from k cat and K m values in amino‐acylation of cognate tRNA Ile ‐C‐C‐A. Factors D are also products of initial discrimination factors I′ and proof‐reading factors II′; D = I′ II′ . Factors II′ were calculated from AMP formation stoichiometries and factors I′ as quotients of D and II′ ; I′ = D/II′ . II′ is considered as a product of a pre‐and post‐transfer proof‐reading factor ( II′ = II 1 II 2 ), I′ as a product of initial discrimination factors I 1 and I 2 which are due to two steps of initial discrimination. With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine ( D = 38000); other D values in a high range (10000–20000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300–700); the remaining amino acids are discriminated with medium D values (1000–10000). Discrimination factors D observed for isoleucyl‐tRNA synthetase from E. coli are on average 2–3 times higher than for the yeast enzyme. Highest values were found in discrimination against Gly, Ala and Val (60000–72000), the lowest values for Cys, Arg and Trp (600–3000); the other amino acids exhibit D values between 20000 and 50000. Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.