Structure—function analysis of the α5 and the α13 helices of human glucokinase: Description of two novel activating mutations

Abstract

It was recently described that the a5 and the a13 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 a5 or the a13 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.

Intragenic Suppression of Gal3C Interaction With Gal80 in the Saccharomyces cerevisiae GAL Gene Switch

“…The binding of glucose and ATP to the active site of the glucokinase forms the ligand-bound active enzyme (Kamata et al 2004). Missense mutations that occur at positions distributed throughout the sequence but colocalize to a hinge region cause the protein to partially adopt the ligandbound state with high enzymatic activities (Glaser et al 1998;Christesen et al 2002;Gloyn 2003;CuestaMunoz et al 2004;Pedelini et al 2005). The binding of a small molecule at this hinge region also activated the enzyme (Grimsby et al 2003), further supporting the idea of allosterism via a hinge-regulated activity.…”

Mutations Distal to the Substrate Site Can Affect Varicella Zoster Virus Thymidine Kinase Activity: Implications for Drug Design

“…These mutations showed that the interactions at the dimer interface are important for the binding of thymidine but not for the catalysis rate, which was increased. Conformational changes affecting catalytic function or inhibitor binding by mutations distal to the active site have been reported in other systems, such as glucokinase and reverse transcriptase (Ren et al, 1998;Pedelini et al, 2005). These observations may be explained by the fact that the VZVTK active site residues (Tyr66 and Gln95) after the loop could have more flexible positions, destabilizing the interactions with thymidine.…”

Biochemical characterization of novel glucokinase mutations isolated from Spanish maturity-onset diabetes of the young (MODY2) patients

“…All the mutants used in this study were obtained as described (Pedelini et al 2005) using the QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA) and the oligonucleotides described in Table 1.…”

“…The modified codon is underlined Name Sequence (2008) 53:460-466 461 (Pedelini et al 2005). The kinetic parameters of the recombinant GST-GlkB proteins were also measured as described (Pedelini et al 2005).…”

“…Upon binding to substrates [glucose and adenosine triphosphate (ATP)], GlkB undergoes a conformational change that brings the large and small domains physically closer, resulting in a closed, active conformation. This structural model has helped in the understanding of the abnormal biochemistry of different GCK missense mutations (Pedelini et al 2005;Galan et al 2006).…”