Table 1.

WT and mutant Cc ProXp-ala deacylation rate constants and relative Ec microhelix^Pro binding affinity

Mutation	Location	Deacylation kobs, min−1*	Fold decrease	Relative binding†
WT		1.05 ± 0.07	1	+++
H23A	β1-α2	0.086 ± 0.019	12	+
P24A	β1-α2	1.07 ± 0.07	0	+++
P25A	β1-α2	0.75 ± 0.11	1.4	+++
F27A	β1-α2	0.35 ± 0.04	3.0	++
R28A	β1-α2	0.30 ± 0.04	3.5	++
V29A	α2	0.53 ± 0.11	2.0	+++
E30A	α2	2.2 ± 0.2	0	+++
E31A	α2	0.29 ± 0.04	3.6	+++
E34A	α2	0.54 ± 0.05	2.0	+
H43A	β2	0.13 ± 0.02	8.2	++
K45A	β2	ND	>100	−
N46S	β2	0.076 ± 0.007	14	++
G98A	α4-α5	0.011 ± 0.002	95	++
S99A	α4-α5	0.39 ± 0.10	2.7	++
V127A	β6	0.50 ± 0.06	2.1	++
N128A	β6	0.17 ± 0.01	6.3	+
H130A	β6-β7	0.11 ± 0.03	9.7	+
N134A	β6-β7	0.098 ± 0.02	11	+++

ND = not detected.

^*Deacylation rate constants (k_obs) were determined using 0.75 μM enzyme and 0.1 μM Ala-tRNA^Pro at 20 °C. All results are an average of three trials with the SD indicated.

^†AUC sedimentation velocity experiments were used to determine the relative binding affinity of microhelix^Pro to mutant ProXp-ala, as described in Fig. S1 B and C. Binding relative to WT ProXp-ala is indicated as follows: +++, less than twofold reduction; ++, twofold to eightfold reduction; +, greater than eightfold reduction; −, no observed binding.