1. 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

2. 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, 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)

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

4. 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

5. 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

6. 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

7. 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