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Acetohydroxyacid synthases (AHAS) and glyoxylate carboligase (GCL) catalyze decarboxylation of 2-ketoacids and condensation of the resulting hydroxyalkylThDP anions/enamines with a second ketoacid to form 2-acyl-2-hydroxyacids. AHASs prefer pyruvate by >10-fold over any other ketoacid as first substrate. Steric hindrance seems to be the major determinant of this specificity; Escherichia coli AHAS isozyme II mutant Val375Ala allows 2-ketobutyrate (C₂H₅COCO₂⁻) to be a good first substrate and the mutant enzyme can thus synthesize 2-propio-2-hydroxybutyrate. An Ile residue in the equivalent position in GCL (Ile393) may play the analogous role in restricting GCL to glyoxylate (HCOCO₂⁻) as first substrate. The specificity of AHAS for 2-ketoacids as acceptor substrates is due to an arginine residue which probably interacts with the carboxylate of the second substrate (e.g., Arg276 in AHASII). Mutants …