TABLE 5.

The effect of selective mutation on GalAK and GalK activities

Selected amino acids of wild-type GalA and Gal proteins were altered by site-directed mutagenesis (“Experimental Procedures”). The recombinant proteins (wt and mutants) were purified and GalAK and GalK activities were measured under standard conditions but for 15 min. The relative activities of mutant were compared to wt (100%), and the value is the mean of duplicate reactions, and the values varied by no more than ±5%.

Enzyme mutation site	Sugar substrate	Relative enzyme activity
		%
GalAKWT	d-GalA	100
	d-GlcA	0
GalAKA41E	d-GalA	0
	d-GlcA	0
GalAKY250F	d-GalA	90
	d-GlcA	50
GalAKA368S	d-GalA	90
	d-GlcA	0
GalKWT	d-Gal	100
	2-d-Gal	100
GalKE62A	d-Gal	0
	2-d-Gal	0
GalKY262F	d-Gal	80
	2-d-Gal	57
GalKA437S	d-Gal	60
	2-d-Gal	40
GalKS206G	d-Gal	95
	2-d-Gal	85
	GalNac	30