1bh5
From Proteopedia
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==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. | ||
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| + | ==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== | ||
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[[Category: lyase]] | [[Category: lyase]] | ||
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov | + | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 16:09:30 2007'' |
Revision as of 14:03, 12 November 2007
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HUMAN GLYOXALASE I Q33E, E172Q DOUBLE MUTANT
Contents |
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.
Disease
Known disease associated with this structure: Autism, susceptibility to OMIM:[138750]
About this Structure
1BH5 is a Single protein structure of sequence from Homo sapiens with ZN and GTX as ligands. Active as Lactoylglutathione lyase, with EC number 4.4.1.5 Structure known Active Sites: GH1, GH2, GH3, GH4, HD2, HD3, HD4, HD5, ZN1, ZN2, ZN3 and ZN4. 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
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