Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing XXXX in humans.

@entity2175 @entity6 @entity72690 @entity36575 @entity14224 @entity1717 @entity72691 @entity164 @entity72689 @entity2596 @entity413 @entity855
@entity164 2 @entity6 is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase ( @entity14224 ). @entity14224 activity is fundamental to beta cell and hepatocyte @entity413 metabolism, and heterozygous activating and inactivating @entity14224 mutations cause @entity1717 ( @entity1717 ) and @entity2175 ( @entity2175 ) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel @entity14224 @entity1717 mutations ( @entity72689 and @entity72690 ) at residues where @entity2175 mutations also occur ( @entity72691 and @entity36575 ). Using recombinant proteins with in vitro assays, we demonstrated that both @entity1717 mutants had a greater relative activity index than wild type (6.0 for @entity72689 , 8.4 for @entity72690 , and 1.0 for wild type). This was driven by an increased affinity for @entity413 (S(0.5), 3.3    0.1 and 3.5    0.1 mm, respectively) versus wild type (7.5    0.1 mm). Correspondingly, the @entity72691 and @entity36575 @entity2175 mutants had markedly reduced relative activity indexes (<0.1). @entity36575 had an altered affinity for @entity413 (S(0.5), 24.9    0.6 mm), whereas @entity72691 also exhibited a reduced affinity for @entity855 and decreased catalysis rate (S(0.5), 78.6    4.5 mm; @entity855 (K(m)), 1.5    0.1 mm; K(cat), 10.3    1.1s(-1)) compared with wild type ( @entity855 (K(m)), 0.4    <0.1; K(cat), 62.9    1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas @entity2596 -389 is located remotely to this position and all other previously reported activating mutations, highlighting a-helix 11 as a novel region regulating @entity14224 activity. Our data suggest that pharmacological manipulation of @entity14224 activity at locations distal from the allosteric activator site is possible.
