. Author manuscript; available in PMC: 2016 Dec 15.

Published in final edited form as: Biochemistry. 2015 Dec 2;54(49):7248–7260. doi: 10.1021/acs.biochem.5b01082

Table 3.

Rate constants for “His → Asp” phosphotransfer

Receiver domain	Amino acid at			YPD1-P ➝ yeast RR			MPI ➝ CheY
Receiver domain	D+2	T+1	T+2	k_max^a (s⁻¹)	K_d^a (μM)	k_max/K_d (× 10⁶ M⁻¹s⁻¹)	k_phos/K_S (M⁻¹s⁻¹)
SLN1/CheY_SLN1	Q	A	F	230 ± 130	7.8 ± 5.7	29	1500 ± 76
SSK1/CheY_SSK1	Q	A	S	160 ± 70	2.4 ± 1.9	67	68 ± 12
SKN7/CheY_SKN7	V	G	N	1.4 ± 0.2	1.5 ± 0.5	0.9	< 0.1^b

Values are from ¹⁴.

Phosphorylation with MPI was below the limit of detection by fluorescence quenching. The observed rate of approach to equilibrium is given by k_obs = (k_phos/K_s)[phosphodonor] + k_dephos,water. To detect phosphorylation, the term describing the phosphorylation rate (which changes with phosphodonor concentration) must be at least as large as the term for dephosphorylation (which is invariant). With [MPI] = 25 mM and k_dephos,water for CheY_SKN7 = 0.002 s⁻¹, setting the phosphorylation and dephosphorylation terms equal gives k_phos/K_S = 0.08 M⁻¹ s⁻¹.