X-Ray crystallography shows that the hydroxyl group of Thr-45 in the fermentative alcohol dehydrogenase (ADH1) from Saccharomyces cerevisiae is hydrogen-bonded to the hydroxyl group of the alcohol bound to the catalytic zinc and is part of a proton relay system linked to His-48. The contribution of Thr-45 to catalysis was studied with steady state kinetics of the enzyme with the T45G substitution. Affinities for coenzymes decrease by only 2-4-fold, but the turnover numbers (V/Et) and catalytic efficiencies (V/KmEt) decrease 480-fold and 2900-fold for the oxidation of ethanol and 450-fold and 8400-fold for acetaldehyde reduction, respectively, relative to wild-type enzyme. Binding of NADH appears to require protonation of a group with a pK value of ∼7.4 in wild-type ADH1, but the pK value for T45G ADH1 appears to be less than 5. For wild-type enzyme, the pH dependencies for ethanol oxidation (V1/Et and V1/KbEt) are maximal above pK values of 7.0-7.7 and are attributed to the ionization of the alcohol or water bound to the catalytic zinc facilitated by His-48 in the enzyme-NAD+ complexes. For T45G ADH1, these pK values are shifted to 6.3. The reduction of acetaldehyde (V2/Et and V2/KpEt) modestly increases as the pH increases for wild-type and T45G enzymes. The removal of the hydroxyethyl group of Thr-45 disrupts the connection of the oxygen of ligands bound to the catalytic zinc with the proton relay system and formation of productive catalytic states. The conformational change of the enzyme and the exchange of ligands on the catalytic zinc can also be affected. Assignments of groups responsible for the pK values are discussed in the context of studies on other forms of horse liver and yeast ADHs. The substitutions with Ala-45 and Cys-45 in yeast ADH1 and the homologous substitutions with Ala-48 in horse and human liver ADHs also significantly decrease catalytic efficiency. Threonine or serine residues at this position in alcohol dehydrogenases are highly conserved and contribute substantially to catalysis.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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