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J. Biol. Chem., Vol. 282, Issue 18, 13906-13916, May 4, 2007

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Glucokinase Thermolability and Hepatic Regulatory Protein Binding Are Essential Factors for Predicting the Blood Glucose Phenotype of Missense Mutations^*

Maria F. Pino¹, Kyoung-Ah Kim¹, Kathy D. Shelton, Jill Lindner, Stella Odili^¶, Changhong Li^¶, Heather W. Collins^¶, Masakazu Shiota, Franz M. Matschinsky^¶, and Mark A. Magnuson²

From the Department of Molecular Physiology and Biophysics and Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the ^¶Department of Biochemistry and Biophysics and Diabetes Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

To better understand how glucokinase (GK) missense mutations associated with human glycemic diseases perturb glucose homeostasis, we generated and characterized mice with either an activating (A456V) or inactivating (K414E) mutation in the gk gene. Animals with these mutations exhibited alterations in their blood glucose concentration that were inversely related to the relative activity index of GK. Moreover, the threshold for glucose-stimulated insulin secretion from islets with either the activating or inactivating mutation were left- or right-shifted, respectively. However, we were surprised to find that mice with the activating mutation had markedly reduced amounts of hepatic GK activity. Further studies of bacterially expressed mutant enzymes revealed that GK^A456V is as stable as the wild type enzyme, whereas GK^K414E is thermolabile. However, the ability of GK regulatory protein to inhibit GK^A456V was found to be less than that of the wild type enzyme, a finding consistent with impaired hepatic nuclear localization. Taken together, this study indicates that it is necessary to have knowledge of both thermolability and the interactions of mutant GK enzymes with GK regulatory protein when attempting to predict in vivo glycemic phenotypes based on the measurement of enzyme kinetics.

Received for publication, October 27, 2006 , and in revised form, March 7, 2007.

^* This work was supported by National Institutes of Health Grant DK42502 (to M. A. M.) and DK 22122 (to F. M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Mentor Award from the American Diabetes Association.

² To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Center for Stem Cell Biology, Vanderbilt University School of Medicine, 802 Light Hall, 2215 Garland Ave., Nashville, TN 37232-0225. Tel.: 615-322-7006; Fax: 615-322-6645; E-mail: mark.magnuson{at}vanderbilt.edu.

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