Table 4.

Enhancement of GSH Nucleophilicity by Grx isoforms^a

substrate	Grx isoform	k(−Grx) (µM−1min−1)	k(+Grx) (µ M−1 min−1)	rate enhancement	enhancement due to ΔpKa (4ΔpKa)	additional enhancement of nucleophilicity
GSH	hGrx1	143 ± 8	(2.58 ± 0.8) × 106	(1.85 ± 0.9) × 104	1024	18.1
	hGrx2	140 ± 8	(2.86 ± 0.5) × 105	(2.0 ± 0.3) × 103	256	7.8
Cys–Gly	hGrx1	98.1 ± 9	(1.51 ± 0.03) × 105	(1.54 ± 0.03) × 103	1024	1.5
	hGrx2	98.1 ± 9	(3.11 ± 0.6) × 104	318 ± 59	256	1.2

^aTime-dependent release of [³H]GSSG from [³H]BSA–SSG was assessed as described in Experimental Procedures. Reaction mixtures contained 0.1 M AMPSO buffer (pH 9.5), GSH or cysteinyl glycine (Cys–Gly) (0.25 mM final concentration), and Grx (3–9 nM). Enzyme and substrate concentrations were within the range of linear dependence (determined in separate experiments). Second-order rate constants (M⁻¹ min⁻¹) for deglutathionylation were calculated as described by Srinivasan et al. (17), i.e., rate of GSSG release = k[BSA–SSG][RSH] (for the uncatalyzed reaction) or k[Grx–SSG][RSH] (for the catalyzed reaction). Final GSH and Cys–Gly concentrations at pH 7.5 were calculated according to their pK_a values [GSH = 8.7, Cys–Gly = 8.8 (17)]. Rate constants represent averages of three to seven determinations ± the standard error.