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J. Biol. Chem., Vol. 281, Issue 52, 40201-40207, December 29, 2006

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Biochemical Basis of Glucokinase Activation and the Regulation by Glucokinase Regulatory Protein in Naturally Occurring Mutations^*

From the Department of Biochemical Pharmacology, La Jolla Laboratories, Pfizer Global Research and Development, San Diego, California 92121

Glucokinase (GK) has several known polymorphic activating mutations that increase the enzyme activity by enhancing glucose binding affinity and/or by alleviating the inhibition of glucokinase regulatory protein (GKRP), a key regulator of GK activity in the liver. Kinetic studies were undertaken to better understand the effect of these mutations on the enzyme mechanism of GK activation and GKRP regulation and to relate the enzyme properties to the associated clinical phenotype of hypoglycemia. Similar to wild type GK, the transient kinetics of glucose binding for activating mutations follows a general two-step mechanism, the formation of an enzyme-glucose complex followed by an enzyme conformational change. However, the kinetics for each step differed from wild type GK and could be grouped into specific types of kinetic changes. Mutations T65I, Y214C, and A456V accelerate glucose binding to the apoenzyme form, whereas W99R, Y214C, and V455M facilitate enzyme isomerization to the active form. Mutations that significantly enhance the glucose binding to the apoenzyme also disrupt the protein-protein interaction with GKRP to a large extent, suggesting these mutations may adopt a more compact conformation in the apoenzyme favorable for glucose binding. Y214C is the most active mutation (11-fold increase in ) and exhibits the most severe clinical effects of hypoglycemia. In contrast, moderate activating mutation A456V nearly abolishes the GKRP inhibition (76-fold increase in K_i) but causes only mild hypoglycemia. This suggests that the alteration in GK enzyme activity may have a more profound biological impact than the alleviation of GKRP inhibition.

Received for publication, August 21, 2006 , and in revised form, November 1, 2006.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Biochemical Pharmacology, La Jolla Laboratories, Pfizer Global Research and Development, 10628 Science Ctr. Dr., San Diego, CA 92121. Tel.: 858-526-4922; Fax: 858-526-4240; E-mail: shaoxian.sun{at}pfizer.com.

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