TABLE 1.

Kinetic parameters of T. brucei TryS in different assay systems

The fixed substrate concentrations used in the HEPES system were 2.5 mm ATP, 1 mm GSH, and 20 mm Spd and in the in vivo-like phosphate buffer 2.5 mm ATP, 2 mm GSH, 8 mm Spd, and 0.5 mm Gsp. Inhibition of TryS by T(SH)₂ versus Spd or Gsp was studied at fixed 0.2 mm GSH (for details see the legend of Fig. 3). The kinetic parameters, which are the means of at least two series of experiments ± S.E. were obtained as described under “Experimental Procedures.” ND, not determined.

Kinetic parameters	HEPPS, pH 8.0a	HEPES, pH 8.0		Phosphate, pH 7.0
	25 °C	25 °C	37 °C	37 °C
	μm	μm		μm
Kmapp Spd	38 ± 5	92 ± 25	139 ± 14	687 ± 90
Kmapp Gsp	2.4 ± 0.2	12 ± 2	16 ± 3	32 ± 5
With Spd as substrate
Kmapp GSH	56 ± 11	32 ± 4	45 ± 3	69 ± 5
Kiapp GSH	37 ± 7	143 ± 20	332 ± 22	849 ± 115
Kmapp ATP	7.1 ± 0.4	6.6 ± 0.5	10 ± 2	18 ± 4
kcat (s−1)	2.9 ± 0.4	1.8 ± 0.1	5.2 ± 0.2	2.8 ± 0.1
Kiapp T(SH)2b	ND	ND	ND	228 ± 112
Kiapp T(SH)2c	ND	ND	ND	377 ± 64
With Gsp as substrate
Kmapp GSH	ND	ND	ND	34 ± 12
Kiapp GSH	ND	ND	ND	1085 ± 309
Kmapp ATP	ND	ND	ND	12 ± 3
kcat (s−1)	ND	ND	ND	2.1 ± 0.3
Kiapp T(SH)2d	ND	ND	ND	223 ± 100
Kiapp T(SH)2c	ND	ND	ND	346 ± 25

^a Data are from Oza et al. (7), with 2 mm ATP, 0.1 mm GSH, and 10 mm Spd as fixed substrate concentrations.

^b Apparent uncompetitive inhibitor constant versus the substrate Spd.

^c Apparent uncompetitive inhibitor constant versus the substrate GSH.

^d Apparent uncompetitive inhibitor constant versus the substrate Gsp.