title: Expression and site-directed mutagenesis of hepatic glucokinase.
abstract: Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.
authors.affiliation: Department of Physiology and Biophysics, SUNY, Stony Brook, NY 11794.
authors.forename: A J
authors.initials: AJ
authors.lastname: Lange
authors.forename: L Z
authors.initials: LZ
authors.lastname: Xu
authors.forename: F
authors.initials: F
authors.lastname: Van Poelwijk
authors.forename: K
authors.initials: K
authors.lastname: Lin
authors.forename: D K
authors.initials: DK
authors.lastname: Granner
authors.forename: S J
authors.initials: SJ
authors.lastname: Pilkis
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005941:Glucokinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D005941:Glucokinase