Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity
Glucokinase (GK) is the central player in glucose-stimulated insulin release from pancreatic β-cells, and catalytic activation by α-d-glucose binding has a key regulatory function. Whereas the mechanism of this activation is well understood, on the basis of crystal structures of human GK, there are...
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| Format: | Online |
| Language: | English |
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Blackwell Publishing Ltd
2011
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531626/ |
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pubmed-35316262013-01-04 Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity Molnes, Janne Teigen, Knut Aukrust, Ingvild Bjørkhaug, Lise Søvik, Oddmund Flatmark, Torgeir Njølstad, Pål Rasmus Original Articles Glucokinase (GK) is the central player in glucose-stimulated insulin release from pancreatic β-cells, and catalytic activation by α-d-glucose binding has a key regulatory function. Whereas the mechanism of this activation is well understood, on the basis of crystal structures of human GK, there are no similar structural data on ATP binding to the ligand-free enzyme and how it affects its conformation. Here, we report on a conformational change induced by the binding of adenine nucleotides to human pancreatic GK, as determined by intrinsic tryptophan fluorescence, using the catalytically inactive mutant form T228M to correct for the inner filter effect. Adenosine-5′-(β,γ-imido)triphosphate and ATP bind to the wild-type enzyme with apparent [L]0.5 (ligand concentration at half-maximal effect) values of 0.27 ± 0.02 mm and 0.78 ± 0.14 mm, respectively. The change in protein conformation was further supported by ATP inhibition of the binding of the fluorescent probe 8-anilino-1-naphthalenesulfonate and limited proteolysis by trypsin, and by molecular dynamic simulations. The simulations provide a first insight into the dynamics of the binary complex with ATP, including motion of the flexible surface/active site loop and partial closure of the active site cleft. In the complex, the adenosine moiety is packed between two α-helices and stabilized by hydrogen bonds (with Thr228, Thr332, and Ser336) and hydrophobic interactions (with Val412 and Leu415). Combined with enzyme kinetic analyses, our data indicate that the ATP-induced changes in protein conformation may have implications for the kinetic cooperativity of the enzyme. Blackwell Publishing Ltd 2011-07 2011-05-31 /pmc/articles/PMC3531626/ /pubmed/21569204 http://dx.doi.org/10.1111/j.1742-4658.2011.08160.x Text en © 2011 The Authors Journal compilation © 2011 FEBS http://creativecommons.org/licenses/by/2.5/ Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Molnes, Janne Teigen, Knut Aukrust, Ingvild Bjørkhaug, Lise Søvik, Oddmund Flatmark, Torgeir Njølstad, Pål Rasmus |
| spellingShingle |
Molnes, Janne Teigen, Knut Aukrust, Ingvild Bjørkhaug, Lise Søvik, Oddmund Flatmark, Torgeir Njølstad, Pål Rasmus Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity |
| author_facet |
Molnes, Janne Teigen, Knut Aukrust, Ingvild Bjørkhaug, Lise Søvik, Oddmund Flatmark, Torgeir Njølstad, Pål Rasmus |
| author_sort |
Molnes, Janne |
| title |
Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity |
| title_short |
Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity |
| title_full |
Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity |
| title_fullStr |
Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity |
| title_full_unstemmed |
Binding of ATP at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity |
| title_sort |
binding of atp at the active site of human pancreatic glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity |
| description |
Glucokinase (GK) is the central player in glucose-stimulated insulin release from pancreatic β-cells, and catalytic activation by α-d-glucose binding has a key regulatory function. Whereas the mechanism of this activation is well understood, on the basis of crystal structures of human GK, there are no similar structural data on ATP binding to the ligand-free enzyme and how it affects its conformation. Here, we report on a conformational change induced by the binding of adenine nucleotides to human pancreatic GK, as determined by intrinsic tryptophan fluorescence, using the catalytically inactive mutant form T228M to correct for the inner filter effect. Adenosine-5′-(β,γ-imido)triphosphate and ATP bind to the wild-type enzyme with apparent [L]0.5 (ligand concentration at half-maximal effect) values of 0.27 ± 0.02 mm and 0.78 ± 0.14 mm, respectively. The change in protein conformation was further supported by ATP inhibition of the binding of the fluorescent probe 8-anilino-1-naphthalenesulfonate and limited proteolysis by trypsin, and by molecular dynamic simulations. The simulations provide a first insight into the dynamics of the binary complex with ATP, including motion of the flexible surface/active site loop and partial closure of the active site cleft. In the complex, the adenosine moiety is packed between two α-helices and stabilized by hydrogen bonds (with Thr228, Thr332, and Ser336) and hydrophobic interactions (with Val412 and Leu415). Combined with enzyme kinetic analyses, our data indicate that the ATP-induced changes in protein conformation may have implications for the kinetic cooperativity of the enzyme. |
| publisher |
Blackwell Publishing Ltd |
| publishDate |
2011 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531626/ |
| _version_ |
1611943200389332992 |