. Author manuscript; available in PMC: 2009 Aug 20.

Published in final edited form as: Chem Rev. 2006 Aug;106(8):3443–3467. doi: 10.1021/cr050317n

Table 1.

Combined energetic effects of enzyme mutagenesis and substrate thio substitution at nonbridging phosphoester oxygens (kcal/mol)

System	ΔΔG^bind^a	ΔG_c^bind^b	ΔΔG^chem	ΔG_c^chem	Ref	System	ΔΔG^chem	ΔG_c^chem	Ref
RNA-Protein					66	UDG			126
R_p	−0.7 to 1.0					WT
S_p	−0.5 to 0.7					+1 R_p	−1.3
						+1 S_p	−2.5
EcoRI					54, 70	−1 R_p	−0.6
						−1 S_p	−0.7
Clamp						−2 R_p	−2.1
R_p	−0.7		−0.1			−2 S_p	+0.5
S_p	−0.9		−0.7
Central						S88A
R_p	+0.3		−0.4			+1 R_p	−1.7	+0.4
S_p	−1.7		+0.2			+1 S_p	−0.5	−2.0
						−1 R_p	−1.6	+1.0
EcoRV					72	−1 S_p	−1.8	+1.1
R_p	0.5 to 6.1
S_p	1.3 to 10					S189A
						+1 R_p	−1.2	−0.1
vTopo					84, 102	+1 S_p	−0.8	−1.7
						−1 R_p	−0.4	−0.1
WT						−1 S_p	−0.6	−0.1
R_p	−1.7		−3.7
S_p	−0.6		−2.6			Phospholipase-C			131–134
R130A
R_p	−1.4	−0.3	−1.3	−2.4		WT
S_p	0	−0.6	+0.3	−2.9		R_p	−2.2
R130K						S_p	−7.5
R_p	−1.3	−0.4	−1.4	−2.3		D33N
S_p	−1.0	+0.4	0	−2.6		R_p	−1.0	−1.2
K167A						S_p	−3.0	−4.5
R_p	−0.7	−1.0	−3.1	−0.6		D33A
S_p	+0.4	−1.0	−3.0	+0.4		R_p	−2.7	+0.5
R223A						S_p	−3.7	−3.8
R_p	−1.4	−0.3	−1.6	−2.1		R69K
S_p	0	−0.6	−1.9	−0.7		R_p	−2.1	−0.1
H265A						S_p	−2.0	−5.5
R_p			−2.1	−1.6
S_p			−2.8	+0.2		Protein Tyrosine Phosphatase			44
RNAse T1					79	WT
						P_s	−5.0
WT						W354A
R_p			−6.7			P_s	−4.5	−0.5
S_p			−0.2			D356A
Y38F						P_s	−5.4	+0.4
R_p			−6.2	−0.5		R409K
S_p			+1.6	−1.8		P_s	−3.2	−1.8
H40A						Q446A
R_p						P_s	−3.8	−1.2
S_p			−1.0	+0.8		Q450A
E58A						P_s	−2.7	−2.3
R_p			−0.8	− 5.9
S_p			−1.0	+0.8
H92Q
R_p
S_p			−0.9	+0.7
F100A
R_p			−3.0	−3.7
S_p			−0.9	+0.7

The corresponding free energy changes for the substitution effects were calculated using ΔΔG^bind = −RT ln(K_D^ox/K_D^s) and ΔΔG^chem = −RT ln(k^ox/k^s) for the binding and chemical steps respectively (T = 25°)..

In the examples where both enzyme mutagenesis and phosphorothioate modifications were performed, the coupling energies for the interaction of an enzyme side chain with a nonbridging oxygen were determined using the equations: ΔG_c^bind = ΔΔG_wt^bind − ΔΔG_mut^bind and ΔG_c^chem = ΔΔG_wt^chem − ΔΔG_mut^chem. A negative coupling energy is expected for a direct interaction with an enzyme side chain and a nonbridging oxygen. The coupling energies that correspond to a direct interaction between a nonbridging oxygen and an enzyme side chain, as judged by x-ray crystallography, are shown in bold in the Table.