Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation.
| Autoři: | Feese MD; Central Laboratories for Key Technology 1 - 13-5 Fukuura Kanazawa Yokohama 236, Japan., Faber HR, Bystrom CE, Pettigrew DW, Remington SJ |
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| Zdroj: | Structure (London, England : 1993) [Structure] 1998 Nov 15; Vol. 6 (11), pp. 1407-18. |
| Způsob vydávání: | Journal Article; Research Support, U.S. Gov't, P.H.S. |
| Jazyk: | English |
| Informace o časopise: | Publisher: Cell Press Country of Publication: United States NLM ID: 101087697 Publication Model: Print Cited Medium: Print ISSN: 0969-2126 (Print) Linking ISSN: 09692126 NLM ISO Abbreviation: Structure Subsets: MEDLINE |
| Imprint Name(s): | Publication: 2000- : Cambridge, Mass. : Cell Press Original Publication: London : Current Biology, c1993- |
| Výrazy ze slovníku MeSH: | Escherichia coli/*enzymology , Glycerol Kinase/*metabolism, Alanine/chemistry ; Allosteric Regulation ; Amino Acid Substitution ; Binding Sites ; Crystallography, X-Ray ; Dimerization ; Glycerol Kinase/chemistry ; Protein Conformation ; Threonine/chemistry |
| Abstrakt: | Background: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. Results: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. Conclusions: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation. |
| Grant Information: | GM-49992 United States GM NIGMS NIH HHS; GM42618 United States GM NIGMS NIH HHS |
| Substance Nomenclature: | 2ZD004190S (Threonine) EC 2.7.1.30 (Glycerol Kinase) OF5P57N2ZX (Alanine) |
| Entry Date(s): | Date Created: 19981118 Date Completed: 19990107 Latest Revision: 20190915 |
| Update Code: | 20240829 |
| DOI: | 10.1016/s0969-2126(98)00140-3 |
| PMID: | 9817843 |
| Autor: | Feese MD; Central Laboratories for Key Technology 1 - 13-5 Fukuura Kanazawa Yokohama 236, Japan., Faber HR, Bystrom CE, Pettigrew DW, Remington SJ |
| Jazyk: | angličtina |
| Zdroj: | Structure (London, England : 1993) [Structure] 1998 Nov 15; Vol. 6 (11), pp. 1407-18. |
| DOI: | 10.1016/s0969-2126(98)00140-3 |
| Abstrakt: | Background: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. Results: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. Conclusions: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation. |
| Databáze: | MEDLINE |
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