Structure/function studies of human beta-cell glucokinase. Enzymatic properties of a sequence polymorphism, mutations associated with diabetes, and other site-directed mutants.
Glucokinase plays a key role in the regulation of glucose metabolism in insulin-secreting pancreatic beta-cells and in the liver. Recent studies have shown that mutations in this enzyme can lead to the development of a form of non-insulin-dependent diabetes mellitus that is characterized by an autosomal dominant mode of inheritance and onset during childhood. Here, we report the catalytic properties of five additional missense mutations associated with diabetes (Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, Trp257-->Arg and Lys414-->Glu), one polymorphism present in both normal and diabetic subjects (Asp4-->Asn), and three site-directed mutations (Glu177-->Lys, Glu256-->Ala, and Lys414-->Ala). The Trp257-->Arg mutation generated an enzyme that had an activity that was less than 0.5% of that for native human beta-cell glucokinase. By contrast, the Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, and Lys414-->Glu mutations had a Vmax that was 20-100% of normal but a Km for glucose that was 8-14-fold greater than the native enzyme. There was no effect of the Asp4-->Asn polymorphism or the Glu177-->Lys substitution on glucokinase activity. The Lys414-->Ala substitution had no effect on Vmax but increased the Km for glucose 2-fold and the Glu256-->Ala substitution caused a approximately 200-fold decrease in Vmax. These studies have led to the identification of additional residues involved in glucokinase catalysis and substrate binding.
Takeda
J
J
Howard Hughes Medical Institute, University of Chicago, Illinois 60637.
Gidh-Jain
M
M
Xu
L Z
LZ
Froguel
P
P
Velho
G
G
Vaxillaire
M
M
Cohen
D
D
Shimada
F
F
Makino
H
H
Nishi
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D003920:Diabetes Mellitus / Q000201:enzymology*; D004926:Escherichia coli; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D006593:Hexokinase; D005941:Glucokinase