Table 1.

Site2 alanine scan of hGH to the hGHR ECD2

	kon (M−1 s−1, ×105)	koff (s−1, ×10−4)	Kda (nM)	Kd(Ala)/Kd(wt)
wt-hGH	2.0	7.6	3.8	1
F1A-hGH	0.77	25.6	33	9
P2A-hGH	1.2	22.4	19	5
I4A-hGH	3.3	295	89	23
P5A-hGH	1.1	19.8	18	5
L6A-hGH	2.3	13.8	6.0	2
R8A-hGH	1.4	32.8	23	6
N12A-hGH	2.8	7.4	2.6	0.7
L15A-hGH	1.2	22.1	18	5
R16A-hGH	2.0	96.1	48	13
R19A-hGH	1.6	20.3	13	3
V102A-hGH	1.8	19.6	11	3
Y103A-hGH	2.2	18.6	8.5	2
N109A-hGH	1.7	120	71	19
D116A-hGH	2.0	6.2	3.1	0.8
E119A-hGH	2.6	25.5	9.8	3
L6A,N12A-hGH	3.0	8.1	2.7	0.71 (1)b
L6A,D116A-hGH	1.9	240	126	33 (2)
N12A,D116A-hGH	2.2	290	132	35 (0.6)
R16A,D116A-hGH	2.6	170	65	17 (11)
R19A,D116A-hGH	2.5	270	108	28 (2)
Y103A,D116A-hGH	3.6	110	31	8 (1)
N109A,D116A-hGH	2.2	45.4	21	6 (13)
E119A,D116A-hGH	2.0	253	127	33 (4)
L6A,N12A,D116A-hGH	1.9	510	268	71 (1)
D116N-hGH	2.6	33.4	13	3.68
D116V-hGH	2.5	23.0	9.2	2.68
L6A,D116V-hGH	1.5	220	147	39 (4)
N12A,D116V-hGH	3.1	220	71	19 (1)
R16A,D116V-hGH	2.0	67.3	34	9 (26)
R19A,D116V-hGH	2.0	237	119	31 (6)
Y103A,D116V-hGH	1.9	143	75	20 (4)
N109A,D116V-hGH	1.7	65	38	10 (38)
E119A,D116V-hGH	1.1	160	145	38 (6)

Experiments were done in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% TWEEN-20 at 25°C.

^a Equilibrium dissociation constant: K_d = k_off/k_on.

^b Values in parentheses are the calculated binding additivities based on the single point mutations: [K_d(Ala¹)/K_d(wt)] × [K_d(Ala²)/K_d(wt)].