Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young.
Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.
Gloyn
Anna L
AL
Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LJ, United Kingdom.
Odili
Stella
S
Zelent
Dorothy
D
Buettger
Carol
C
Castleden
Harriet A J
HA
Steele
Anna M
AM
Stride
Amanda
A
Shiota
Chyio
C
Magnuson
Mark A
MA
Lorini
Renata
R
d'Annunzio
Giuseppe
G
Stanley
Charles A
CA
Kwagh
Jae
J
van Schaftingen
Emile
E
Veiga-da-Cunha
Maria
M
Barbetti
Fabrizio
F
Dunten
Pete
P
Han
Yi
Y
Grimsby
Joseph
J
Taub
Rebecca
R
Ellard
Sian
S
Hattersley
Andrew T
AT
Matschinsky
Franz M
FM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002648:Child; D004252:DNA Mutational Analysis; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005260:Female; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000235:genetics / Q000378:metabolism; D007231:Infant, Newborn; D008297:Male; D008958:Models, Molecular; D010375:Pedigree; D017354:Point Mutation; D011247:Pregnancy; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C485100:GCKR protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005941:Glucokinase; D005947:Glucose