Effect of mutations on the sensitivity of human beta-cell glucokinase to liver regulatory protein.
Human beta-cell glucokinase and its liver counterpart displayed a half-saturating concentration of glucose (S0.5) of about 8 mmol/l and a Hill coefficient of 1.7, and were as sensitive to inhibition by the rat liver regulatory protein as the rat liver enzyme. These results indicate that the N-terminal region of glucokinase, which differs among these three enzymes, is not implicated in the recognition of the regulatory protein. They also suggest that the regulatory protein, or a related protein, could modulate the affinity of glucokinase for glucose in beta cells. We have also tested the effect of several mutations, many of which are implicated in maturity onset diabetes of the young. The mutations affected the affinity for glucose and for the regulatory protein to different degrees, indicating that the binding site for these molecules is different. An Asp158 Ala mutation, found in the expression plasmid previously thought to encode the wild-type enzyme, increased the affinity for glucose by about 2.5-fold without changing the affinity for the regulatory protein. The mutations that were found to decrease the affinity for the regulatory protein (Asn166 Arg. Val203 Ala, Asn204 Gln, Lys414 Ala) clustered in the hinge region of glucokinase and nearby in the large and small domains. These results are in agreement with the concept that part of the binding site for the regulatory protein is situated in the hinge region of this enzyme.
Veiga-da-Cunha
M
M
Laboratory of Physiological Chemistry, Institute of Cellular and Molecular Pathology, Brusscls, Belgium.
Xu
L Z
LZ
Lee
Y H
YH
Marotta
D
D
Pilkis
S J
SJ
Van Schaftingen
E
E
D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine; D000818:Animals; D001224:Aspartic Acid; D002352:Carrier Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005941:Glucokinase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012097:Repressor Proteins / Q000494:pharmacology*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; C485100:GCKR protein, human; C499484:Gckr protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C080906:glucokinase regulatory protein; D001224:Aspartic Acid; D005941:Glucokinase; D000409:Alanine