HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: ps.051485205v1 14/8/2080    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pedelini, L. Articles by Sanz, P. Search for Related Content PubMed PubMed Citation Articles by Pedelini, L. Articles by Sanz, P. Social Bookmarking                   What's this?

Structure—function analysis of the 5 and the 13 helices of human glucokinase: Description of two novel activating mutations

¹ Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain
² Fundación Instituto Mediterráneo para el Avance de la Biotecnología y la Investigación Sanitaria (IMABIS), Málaga, Spain
³ Department of Endocrinology, Diabetes and Nutrition, Carlos Haya Hospital, Málaga, Spain

(RECEIVED March 31, 2005; FINAL REVISION May 3, 2005; ACCEPTED May 4, 2005)

It was recently described that the 5 and the 13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site-directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild-type and previously defined activating mutations in these helices (A456V and Y214C). We found two new activating mutations, A460R and Y215A, which increase the affinity of the enzyme for glucose. Our results suggest that substitutions in the 5 or the 13 helices that favor the closed, active conformation of the enzyme, either by improving the interaction with surrounding residues or by improving the flexibility of the region defined by these two helices, enhance the affinity of the enzyme for glucose, and therefore its performance as a glucose phosphorylating enzyme.

Keywords: glucokinase; activating mutation; GKAs; glucose signaling; enzyme kinetics

Abbreviations: GST, glutathione-S-transferase • IPTG, isopropyl--D-thiogalactoside • PCR, polymerase chain reaction • SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051485205.

Reprint requests to: Pascual Sanz, Instituto de Biomedicina de Valencia (CSIC), Jaime Roig 11, 46010-Valencia, Spain; e-mail: sanz{at}ibv.csic.es; fax: +3496-3690800.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L. Bjorkhaug, J. Molnes, O. Sovik, P. R. Njolstad, and T. Flatmark Allosteric Activation of Human Glucokinase by Free Polyubiquitin Chains and Its Ubiquitin-dependent Cotranslational Proteasomal Degradation J. Biol. Chem., August 3, 2007; 282(31): 22757 - 22764. [Abstract] [Full Text] [PDF]

				V. V. Heredia, T. J. Carlson, E. Garcia, and S. Sun Biochemical Basis of Glucokinase Activation and the Regulation by Glucokinase Regulatory Protein in Naturally Occurring Mutations J. Biol. Chem., December 29, 2006; 281(52): 40201 - 40207. [Abstract] [Full Text] [PDF]

				K. El Omari, S. Liekens, L. E. Bird, J. Balzarini, and D. K. Stammers Mutations Distal to the Substrate Site Can Affect Varicella Zoster Virus Thymidine Kinase Activity: Implications for Drug Design Mol. Pharmacol., June 1, 2006; 69(6): 1891 - 1896. [Abstract] [Full Text] [PDF]

				C. Q. Diep, G. Peng, M. Bewley, V. Pilauri, I. Ropson, and J. E. Hopper Intragenic Suppression of Gal3C Interaction With Gal80 in the Saccharomyces cerevisiae GAL Gene Switch Genetics, January 1, 2006; 172(1): 77 - 87. [Abstract] [Full Text] [PDF]