Biochemical characterization of MODY2 glucokinase variants V62M and G72R reveals reduced enzymatic activities relative to wild type.
The glucokinase V62M and G72R mutations are naturally occurring and known to associate with hyperglycemia in humans. Structurally, V62 and G72 residues are located in close proximity to the allosteric site where hypoglycemia-linked activating mutations are clustered. To address the mechanism by which these variants alter the physiological phenotype, we characterized the biochemical and biophysical properties of the enzymes. Recombinant proteins were purified without affinity tags, and their steady-state kinetics and glucose binding affinities were determined. Both enzymes showed reduced rates of turnover (k(cat)) and reduced glucose affinity (i.e., increased K(0.5) and K(D) values). Their thermal stability did not largely differ from that of wild-type glucokinase. However, V62M and G72R lost the stabilizing protein interactions with glucokinase regulatory protein, which may contribute to lower activity in vivo. Both mutants were subject to activation by small molecule activators. In conclusion, the decreased enzyme activities of V62M and G72R observed in this study are consistent with the hyperglycemic phenotype.
Ralph
Erik C
EC
Department of Biochemical Pharmacology, La Jolla Laboratories, Pfizer Global Research and Development, San Diego, California 92121, USA.
Sun
Shaoxian
S
D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D014644:Genetic Variation; D005941:Glucokinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense
D005941:Glucokinase; D005947:Glucose