Chap 13. Specificity and Editing Mechanism The relative rate of reaction A function of both substrate binding and catalytic rate The kcat/KM: the important kinetic constant in determining specificity

A. Limits on Specificity Larger substrates than the specific substrate: small active site to fit in Smaller substrates than the specific substrate: always able to bind, less binding energy Ex. aminoacyl tRNA synthethases isoleucyl-tRNA synthetase: Ile vs Val, 150 times tighter valyl-tRNA synthetase: Val vs. Thr, 100-200 times tighter alanyl-tRNA synthetase: Ala vs Gly, 250 times tighter The maximum effect due to the additional binding: exp(-Gb/RT) (Gb, the binding energy of the enzyme and TS)

B. Editing or Proofreading Mechanisms Proofreading exists In DNA replication the complementary base pairing: 1 part in 104-105 error rate: 1 per 108-1010 In protein biosynthesis amino acid selection: 1 part in 102 error rate: 1 per 103-104 Active site for synthesis and hydrolytic active site

There are Different Editing Mechanisms between Protein Synthesis and DNA Replication each specific enzyme for selecting an amino acid error correction before polymerization DNA replication a single DNA polymerase the editing after polymerization

Three Rules of Proof may be Considered in Protein Synthesis tRNA E + ATP + AA E•AA-AMP AA-tRNA + AMP + E PPi The valyl-tRNA synthetase: the mischarged tRNA Val vs. Thr, 600 times the hydrophobic acylating site and the hydrophilic deacylating site The isoleucyl-tRNA synthetase: the aminoacyl adenylate (E•AA-AMP) with existing tRNA, major editing process The methionyl-tRNA synthetase: the misactivated homocyteine without tRNA, before transfer to tRNA, the cyclized intermediate

Prokaryotic DNA Polymerases have a 3’→5’ Exonuclease Activity to Give the Postreplicative Repair Mechanism DNA synthesis: 5’→ 3’, Exonuclease activity: 3’→ 5’ The exonuclease activity is greatest for mismatched bases or ssDNA The mutation frequency correlates with the exonuclease activity: Table 13.1 Prokaryotic DNA polymerases (with exonuclease activity): higher accuracy Eukaryotic DNA polymerases (no exonuclease activity): expected accuracy The kinetics of error induction by prokaryotic DNA polymerases are consistent with the active participation of an editing mechanism

C. The Cost of Accuracy The editing hydrolysis costs energy: energy wasteful hydrolysis of the correct substrate Cost: the fraction of correct substrate wastefully hydrolyzed The more efficient the editing, the more the cost In general, higher selectivity at lower cost: multistage editing