Table V.

Summary of the analysis of the sulfotransferase activity of 3-OST-3 mutants

Name of the constructsa	Relative activityb	Expression levelc
	%
3-OST-3 wild type	100	+ +
3-OST-3 K161A	0.4	+
3-OST-3 K162A	0.4	+
3-OST-3 R166E	0.2	+ +
3-OST-3 E170Q	83	+ +
3-OST-3 R173S	55.9	+ + +
3-OST-3 G182A	106	+ +
3-OST-3 E184Q	0.1	+ +
3-OST-3 H186F	0.0	+
3-OST-3 D189N	0.9	+
3-OST-3 R190E	68	+ +
3-OST-3 K194A	0.5	+
3-OST-3 K215A	0.1	+ + +
3-OST-3 S218A	76.7	+ +
3-OST-3 E224Q	52.4	+ +
3-OST-3 Q255A	0.4	+ +
3-OST-3 K259A	51.7	+ +
3-OST-3 I288A	35	+ +
3-OST-3 K293A	66.4	+ +
3-OST-3 H362A	101.5	+ +
3-OST-3 G365A	57	+ +
3-OST-3 K366A	0.2	+ +
3-OST-3 K368A	0.1	+ +
3-OST-3 R370E	0.8	+ +

^aThe mutants were prepared using a site-directed mutagenesis kit from Invitrogen.

^bThe activity of 3-OST-3 was assayed by incubating the purified mutant proteins with HS and [³⁵S]PAPS, and the resultant [³⁵S]HS was quantified by a DEAE chromatography, where 100% activity represents the transfer of 18 pmol of sulfate/μg of protein under the standard assay condition.

^cThe expression level of the proteins was determined by the intensity of the Coomassie Blue-stained protein band migrated at 30 kDa on SDS-PAGE.