Table 1.

Ligand dissociation constants for substrate-fragments with APS reductase.

Ligand	Structure	Kd [μM][c]	ΔΔG [kcal/mol][d]	pKa
APS[a]		0.20	N/A	~2 (O)[e]
AMP		5.4	2.0	6.8 (O)52
5′-Phosphoribose		93	5.1	~6.8 (O)[f]
Adenosine		3000	5.8	3.6 (N1), 12.4 (O)54
Phosphate		66000	7.7	1.97 (O), 6.82 (O), 12.5 (O)55
Ribose		680000	9.1	12.22 (O)56
Adenine[b]		≥90000	≥7.3	4.15 (N1), 9.80 (N9)56
Sulfate[b]		≥310000	≥8.6	−3 (O), 1.89 (O)57

^[a]The K_d of APS was measured under single turnover conditions, in the absence of thioredoxin, as described in the methods section.

^[b]Due to the limits of solubility or solution ideality the reported values are lower limits.

^[c]For substrate-fragments in this table values of K_i were determined under single turnover conditions from the dependence of the observed rate constant at a given inhibitor concentration under conditions of subsaturating APS, such that K_i is equal to the K_d. Each value reflects the average of at least two independent experiments, and the standard deviation was less than 15% of the value of the mean. Kinetic data were nonlinear-least squares fit to a model of competitive inhibition.

^[d]Energetic difference in affinity of APS relative to inhibitor, ΔΔG = −RTln(K_d^APS/K_d^Fragment).

^[e]pKa estimated from value measured for 2′-deoxy-5′-phosphoribose⁵⁸.

^[f]pKa estimated from value measured for 3′-phospho-5′-adenosinephosphosulfate⁵⁹.