1bh5
From Proteopedia
| Line 4: | Line 4: | ||
|PDB= 1bh5 |SIZE=350|CAPTION= <scene name='initialview01'>1bh5</scene>, resolution 2.2Å | |PDB= 1bh5 |SIZE=350|CAPTION= <scene name='initialview01'>1bh5</scene>, resolution 2.2Å | ||
|SITE= <scene name='pdbsite=GH1:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH1</scene>, <scene name='pdbsite=GH2:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH2</scene>, <scene name='pdbsite=GH3:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH3</scene>, <scene name='pdbsite=GH4:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH4</scene>, <scene name='pdbsite=HD2:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD2</scene>, <scene name='pdbsite=HD3:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD3</scene>, <scene name='pdbsite=HD4:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD4</scene>, <scene name='pdbsite=HD5:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD5</scene>, <scene name='pdbsite=ZN1:Zn+Binding+Site+At+Dimer+Interface'>ZN1</scene>, <scene name='pdbsite=ZN2:Zn+Binding+Site+At+Dimer+Interface'>ZN2</scene>, <scene name='pdbsite=ZN3:Zn+Binding+Site+At+Dimer+Interface'>ZN3</scene> and <scene name='pdbsite=ZN4:Zn+Binding+Site+At+Dimer+Interface'>ZN4</scene> | |SITE= <scene name='pdbsite=GH1:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH1</scene>, <scene name='pdbsite=GH2:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH2</scene>, <scene name='pdbsite=GH3:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH3</scene>, <scene name='pdbsite=GH4:Binding+Site+For+Gsh+Moiety+-+Substrate+Binding+Site+Is+...'>GH4</scene>, <scene name='pdbsite=HD2:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD2</scene>, <scene name='pdbsite=HD3:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD3</scene>, <scene name='pdbsite=HD4:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD4</scene>, <scene name='pdbsite=HD5:Hydrophobic+Substrate+Binding+Pocket+At+Dimer+Interface'>HD5</scene>, <scene name='pdbsite=ZN1:Zn+Binding+Site+At+Dimer+Interface'>ZN1</scene>, <scene name='pdbsite=ZN2:Zn+Binding+Site+At+Dimer+Interface'>ZN2</scene>, <scene name='pdbsite=ZN3:Zn+Binding+Site+At+Dimer+Interface'>ZN3</scene> and <scene name='pdbsite=ZN4:Zn+Binding+Site+At+Dimer+Interface'>ZN4</scene> | ||
| - | |LIGAND= <scene name='pdbligand= | + | |LIGAND= <scene name='pdbligand=GTX:S-HEXYLGLUTATHIONE'>GTX</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</scene> |
| - | |ACTIVITY= [http://en.wikipedia.org/wiki/Lactoylglutathione_lyase Lactoylglutathione lyase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.4.1.5 4.4.1.5] | + | |ACTIVITY= <span class='plainlinks'>[http://en.wikipedia.org/wiki/Lactoylglutathione_lyase Lactoylglutathione lyase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.4.1.5 4.4.1.5] </span> |
|GENE= | |GENE= | ||
| + | |DOMAIN= | ||
| + | |RELATEDENTRY= | ||
| + | |RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1bh5 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1bh5 OCA], [http://www.ebi.ac.uk/pdbsum/1bh5 PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1bh5 RCSB]</span> | ||
}} | }} | ||
| Line 14: | Line 17: | ||
==Overview== | ==Overview== | ||
The Zn2+ ligands glutamate 99 and glutamate 172 in the active site of human glyoxalase I were replaced, each in turn, by glutamines by site-directed mutagenesis to elucidate their potential significance for the catalytic properties of the enzyme. To compensate for the loss of the charged amino acid residue, another of the metal ligands, glutamine 33, was simultaneously mutated into glutamate. The double mutants and the single mutants Q33E, E99Q, and E172Q were expressed in Escherichia coli, purified on an S-hexylglutathione matrix, and characterized. Metal analysis demonstrated that mutant Q33E/E172Q contained 1.0 mol of zinc/mol of enzyme subunit, whereas mutant Q33E/E99Q contained only 0.3 mol of zinc/mol of subunit. No catalytic activity could be detected with the double mutant Q33E/E172Q (<10(-8) of the wild-type activity). The second double mutant Q33E/E99Q had 1.5% of the specific activity of the wild-type enzyme, whereas the values for mutants Q33E and E99Q were 1.3 and 0. 1%, respectively; the E172Q mutant had less than 10(-5) times the specific activity of the wild-type. The crystal structure of the catalytically inactive double mutant Q33E/E172Q demonstrated that Zn2+ was bound without any gross changes or perturbations. The results suggest that the metal ligand glutamate 172 is directly involved in the catalytic mechanism of the enzyme, presumably serving as the base that abstracts a proton from the hemithioacetal substrate. | The Zn2+ ligands glutamate 99 and glutamate 172 in the active site of human glyoxalase I were replaced, each in turn, by glutamines by site-directed mutagenesis to elucidate their potential significance for the catalytic properties of the enzyme. To compensate for the loss of the charged amino acid residue, another of the metal ligands, glutamine 33, was simultaneously mutated into glutamate. The double mutants and the single mutants Q33E, E99Q, and E172Q were expressed in Escherichia coli, purified on an S-hexylglutathione matrix, and characterized. Metal analysis demonstrated that mutant Q33E/E172Q contained 1.0 mol of zinc/mol of enzyme subunit, whereas mutant Q33E/E99Q contained only 0.3 mol of zinc/mol of subunit. No catalytic activity could be detected with the double mutant Q33E/E172Q (<10(-8) of the wild-type activity). The second double mutant Q33E/E99Q had 1.5% of the specific activity of the wild-type enzyme, whereas the values for mutants Q33E and E99Q were 1.3 and 0. 1%, respectively; the E172Q mutant had less than 10(-5) times the specific activity of the wild-type. The crystal structure of the catalytically inactive double mutant Q33E/E172Q demonstrated that Zn2+ was bound without any gross changes or perturbations. The results suggest that the metal ligand glutamate 172 is directly involved in the catalytic mechanism of the enzyme, presumably serving as the base that abstracts a proton from the hemithioacetal substrate. | ||
| - | |||
| - | ==Disease== | ||
| - | Known disease associated with this structure: Autism, susceptibility to OMIM:[[http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=138750 138750]] | ||
==About this Structure== | ==About this Structure== | ||
| Line 28: | Line 28: | ||
[[Category: Cameron, A D.]] | [[Category: Cameron, A D.]] | ||
[[Category: Jones, T A.]] | [[Category: Jones, T A.]] | ||
| - | [[Category: GTX]] | ||
| - | [[Category: ZN]] | ||
[[Category: glyoxalase i]] | [[Category: glyoxalase i]] | ||
[[Category: lactoylglutathione lyase]] | [[Category: lactoylglutathione lyase]] | ||
[[Category: lyase]] | [[Category: lyase]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Mar 30 19:00:55 2008'' |
Revision as of 16:00, 30 March 2008
| |||||||
| , resolution 2.2Å | |||||||
|---|---|---|---|---|---|---|---|
| Sites: | , , , , , , , , , , and | ||||||
| Ligands: | , | ||||||
| Activity: | Lactoylglutathione lyase, with EC number 4.4.1.5 | ||||||
| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
HUMAN GLYOXALASE I Q33E, E172Q DOUBLE MUTANT
Overview
The Zn2+ ligands glutamate 99 and glutamate 172 in the active site of human glyoxalase I were replaced, each in turn, by glutamines by site-directed mutagenesis to elucidate their potential significance for the catalytic properties of the enzyme. To compensate for the loss of the charged amino acid residue, another of the metal ligands, glutamine 33, was simultaneously mutated into glutamate. The double mutants and the single mutants Q33E, E99Q, and E172Q were expressed in Escherichia coli, purified on an S-hexylglutathione matrix, and characterized. Metal analysis demonstrated that mutant Q33E/E172Q contained 1.0 mol of zinc/mol of enzyme subunit, whereas mutant Q33E/E99Q contained only 0.3 mol of zinc/mol of subunit. No catalytic activity could be detected with the double mutant Q33E/E172Q (<10(-8) of the wild-type activity). The second double mutant Q33E/E99Q had 1.5% of the specific activity of the wild-type enzyme, whereas the values for mutants Q33E and E99Q were 1.3 and 0. 1%, respectively; the E172Q mutant had less than 10(-5) times the specific activity of the wild-type. The crystal structure of the catalytically inactive double mutant Q33E/E172Q demonstrated that Zn2+ was bound without any gross changes or perturbations. The results suggest that the metal ligand glutamate 172 is directly involved in the catalytic mechanism of the enzyme, presumably serving as the base that abstracts a proton from the hemithioacetal substrate.
About this Structure
1BH5 is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
Reference
Involvement of an active-site Zn2+ ligand in the catalytic mechanism of human glyoxalase I., Ridderstrom M, Cameron AD, Jones TA, Mannervik B, J Biol Chem. 1998 Aug 21;273(34):21623-8. PMID:9705294
Page seeded by OCA on Sun Mar 30 19:00:55 2008
