GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK–glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK–glucose–GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (kcat, glucose S0.5, h and ATP Km), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S0.5, but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.
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Research Article|
November 14 2011
Mutational analysis of allosteric activation and inhibition of glucokinase
Bogumil Zelent;
Bogumil Zelent
*Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, 415 Curie Blvd, 700 CRB, Philadelphia, PA 19104, U.S.A.
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Stella Odili;
Stella Odili
*Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, 415 Curie Blvd, 700 CRB, Philadelphia, PA 19104, U.S.A.
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Carol Buettger;
Carol Buettger
*Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, 415 Curie Blvd, 700 CRB, Philadelphia, PA 19104, U.S.A.
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Dorothy K. Zelent;
Dorothy K. Zelent
*Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, 415 Curie Blvd, 700 CRB, Philadelphia, PA 19104, U.S.A.
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Pan Chen;
Pan Chen
†Division of Endocrinology, The Children's Hospital of Philadelphia, 34th Street and Civic Center Blvd., Philadelphia, PA 19104, U.S.A.
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Deborah Fenner;
Deborah Fenner
‡Department of Medicine, Feinberg School of Medicine, Northwestern University, Evanston, IL 60208, U.S.A.
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Joseph Bass;
Joseph Bass
‡Department of Medicine, Feinberg School of Medicine, Northwestern University, Evanston, IL 60208, U.S.A.
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Charles Stanley;
Charles Stanley
†Division of Endocrinology, The Children's Hospital of Philadelphia, 34th Street and Civic Center Blvd., Philadelphia, PA 19104, U.S.A.
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Monique Laberge;
Monique Laberge
§Royal Military College St-Jean, St-Jean, QC J0J 1R0, Canada
¶Concordia University, Department of Biology, Montréal, QC H3G 1M8, Canada
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Jane M. Vanderkooi;
Jane M. Vanderkooi
*Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, 415 Curie Blvd, 700 CRB, Philadelphia, PA 19104, U.S.A.
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Ramakanth Sarabu;
Ramakanth Sarabu
∥Departments of Discovery Chemistry and Metabolic Diseases, Hoffmann-La Roche, Nutley, NJ 07110, U.S.A.
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Joseph Grimsby;
Joseph Grimsby
∥Departments of Discovery Chemistry and Metabolic Diseases, Hoffmann-La Roche, Nutley, NJ 07110, U.S.A.
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Franz M. Matschinsky
Franz M. Matschinsky
1
*Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, 415 Curie Blvd, 700 CRB, Philadelphia, PA 19104, U.S.A.
1To whom correspondence should be addressed (email matsch@mail.med.upenn.edu).
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Publisher: Portland Press Ltd
Received:
March 08 2011
Revision Received:
July 27 2011
Accepted:
August 10 2011
Accepted Manuscript online:
August 10 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 440 (2): 203–215.
Article history
Received:
March 08 2011
Revision Received:
July 27 2011
Accepted:
August 10 2011
Accepted Manuscript online:
August 10 2011
Citation
Bogumil Zelent, Stella Odili, Carol Buettger, Dorothy K. Zelent, Pan Chen, Deborah Fenner, Joseph Bass, Charles Stanley, Monique Laberge, Jane M. Vanderkooi, Ramakanth Sarabu, Joseph Grimsby, Franz M. Matschinsky; Mutational analysis of allosteric activation and inhibition of glucokinase. Biochem J 1 December 2011; 440 (2): 203–215. doi: https://doi.org/10.1042/BJ20110440
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