Table 2.

Effects of Group-Selective Reagents on STR1 Activity

	[Reagent] (mM)	[Reagent]/[STR1] (mol/mol)	Group Selectivitya	Inactivation (%)
Reagent				30 min	2 h	Buffer
DCC	0.05	50	Asp/Glu	100		A
WK	1.00	1,000	Asp/Glu	48	53	A
	5.00	5,000		80	100
DEPC	1.00	1,000	His	49	63	B
PCMB	0.20	200	Cys	34	35	B
PMSF	1.00	1,000	Ser/Cys	0	0	C
TPCK	0.12	120	Ser/Cys	0	0	C
TLCK	0.25	250	Ser/Cys	0	0	C
AEBSF	4.00	4,000	Ser/Cys	4	12	C
NAI	10.00	10,000	Tyr	5	12	D
DNFB	5.00	5,000	Lys/Arg	0	15	D

Selective modification of reactive residues of STR1 was performed by adding various amounts (0.05 to 10 mM) of reagents to 1 μM enzyme solution in appropriate buffers. Aliquots of incubation mixture were taken at time intervals, and STR1 activity was measured. The pH of the reaction as well as the molar excess of reagent over the enzyme was designed to optimize the group selectivity of each reagent. DCC, N,N′-dicyclohexylcarbodiimide; WK, N-ethyl-5-phenylisoxazolium-3′-sulfonate (Woodward's reagent K); DEPC, diethylpyrocarbonate; PCMB, p-chloromercuribenzoate; PMSF, phenylmethanesulfonyl fluoride; TPCK, l-chloro-3-(4-tosyl-amido)-4-phenyl-2-butanone; TLCK, l-chloro-3-(4-tosyl-amido)-7-amino-2-heptanone; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl-fluoride; NAI, N-acetylimidazole; DNFB, dinitrofluorobenzene. Buffer A, 50 mM MES, pH 6.0; buffer B, 100 mM potassium phosphate buffer, pH 6.0; buffer C, 100 mM potassium phosphate buffer, pH 7.0; buffer D, 10 mM Tris-HCl, pH 8.0.

^aThe most likely side chains modified under the conditions of the experiment.