Structural Basis for Allosteric Regulation of the Monomeric Allosteric Enzyme Human Glucokinase

Kamata, Kenji; Mitsuya, Morihiro; Nishimura, Teruyuki; Eiki, Jun-ichi; Nagata, Yasufumi

doi:10.1016/j.str.2004.02.005

Cited by 446 publications

(737 citation statements)

References 45 publications

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“…Loss of regulation by GCKR and allosteric activators has been reported for other GCK mutants with increased or nearly-normal enzyme activity [18,31]. Despite this, we found that the R397L mutation did not affect GCK-GCKR binding in vitro and was not located in the allosteric site predicted from the crystal structure [30]. A possible effect of this mutation on the interaction of GCK with other intracellular partners has been investigated.…”

Section: Discussionsupporting

confidence: 44%

“…However, and in contrast to M235V, M235T also resulted in a decrease in the K m for ATP and in the affinity for glucose. Interestingly, the crystal structure of GCK complexed with a GCK allosteric activator indicates that the M235 residue, together with residues V62, I159, M210, I211 and V452, is involved in hydrophobic interactions with this compound [30]. As expected from this model, MODY2 mutations V62M and M210K result in a loss of effect of GCK activators as well as a loss of inhibition by GCKR [18,31].…”

Section: Discussionmentioning

confidence: 67%

“…3 Localisation of residues D160, K161, M235, R308 and R397 on the structural model for beta cell GCK. The closed conformation (1V4S) of wild-type GCK [30] is represented using the RasMol program [36]. Selected residues are indicated as clusters of coloured balls.…”

Section: Discussionmentioning

confidence: 99%

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Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

et al. 2006

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Aims/hypothesis Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. Materials and methods Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested. Results We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR. Conclusions/interpretation Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity.

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Section: Discussionsupporting

confidence: 44%

Section: Discussionmentioning

confidence: 67%

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Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

et al. 2006

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“…2 and 3). The blunted GSIS could be due to reduced GK expression (24) and adoption of its "wide-open" conformation with low activity and resistance to GKA effect (15,20). Thus, although Ro significantly increased the glucose sensitivity of islets cultured at G10 (Fig.…”

Section: Discussionmentioning

confidence: 95%

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration

Roma

Duprez

Jonas³

2015

American Journal of Physiology-Endocrinology and Metabolism

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Roma LP, Duprez J, Jonas JC. Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration. Am J Physiol Endocrinol Metab 309: E632-E639, 2015. First published August 11, 2015; doi:10.1152/ajpendo.00154.2015.-In rat pancreatic islets, ␤-cell gene expression, survival, and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here, we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. Rat pancreatic islets were cultured 1-7 days at G5, G10, or G30 with or without 3 M of the GK activator Ro 28-0450 (Ro). After culture, ␤-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H autofluorescence, intracellular calcium concentration, and insulin secretion were tested in the absence or presence of Ro. Prolonged culture of rat islets at G5 or G30 instead of G10 triggered ␤-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected ␤-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to ␤-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10 but failed at restoring ␤-cell glucose responsiveness after culture at G5 or G30. We conclude that pharmacological GK activation prevents the alteration of ␤-cell survival and function by long-term culture at G5 but mimics glucotoxicity when added to G10. The complex effects of glucose on the ␤-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se. apoptosis; glucotoxicity; insulin secretion; pancreatic ␤-cell GLUCOSE STIMULATION OF INSULIN SECRETION (GSIS) by endocrine pancreatic ␤-cells depends on the acceleration of glucose metabolism through glycolysis and the TCA cycle, with enhanced production of metabolic coupling factors (25,39,41). Besides these rapid effects, glucose exerts complex long-term effects on the ␤-cell phenotype (2,10,16,18,27,45). During long-term culture of rodent islets, ␤-cell gene expression, survival, and glucose responsiveness are optimally preserved in the presence of 10 mM glucose (G10), whereas they are markedly altered by culture at either nonstimulating (G5) or very high (G30) glucose concentrations. In other words, culture at G10 vs. G5 is beneficial, whereas culture at G30 vs. G10 is detrimental to ␤-cell gene expression, survival, and glucose responsiveness. The beneficial effect of culture at G10 vs. G5 is usually attributed to the stimulation of energetic metabolism. In contrast, the deleterious effects of culture at G30 vs. G10, which we later refer to as glucotoxicity, could result from the further increase in metabolism with increased production o...

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“…In 2004, Katama and colleagues determined the crystal structures of human GK in both active and inactive forms [12]. Their results showed that GK has a small domain and a large domain separated by a deep cleft and undergoes a large conformational change by the rotation of the small domain induced by glucose binding.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of E339K mutated human glucokinase reveals changes in the ATP binding site

et al. 2011

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a b s t r a c tHuman glucokinase (GK) plays an important role in glucose homeostasis. An E339K mutation in GK was recently found to be associated with hyperglycemia. It showed lower enzyme activity and impaired protein stability compared to the wild-type enzyme. Here, we present the crystal structure of E339K GK in complex with glucose. This mutation results in a conformational change of His416, spatially interfering with adenosine-triphosphate (ATP) binding. Furthermore, Ser411 at the ATP binding site is phosphorylated and then hydrogen bonded with Thr82, physically blocking the ATP binding. These findings provide structural basis for the reduced activity of this mutant.

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Structural Basis for Allosteric Regulation of the Monomeric Allosteric Enzyme Human Glucokinase

Cited by 446 publications

References 45 publications

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration

Crystal structure of E339K mutated human glucokinase reveals changes in the ATP binding site

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