Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.
The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The two Arabidopsis thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coli and purified to homogeneity. Biochemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reaction than TyrAAT2. Moreover, TyrAAT2 presents a weak prephenate dehydrogenase activity whereas TyrAAT1 does not. The mechanism of the dehydrogenase reaction catalyzed by these two forms has been investigated using steady-state kinetics. For both enzymes, steady-state velocity patterns are consistent with a rapid equilibrium, random mechanism in which two dead-end complexes, E-NADPH-arogenate and E-NADP-tyrosine, are formed.
D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic / Q000378:metabolism / Q000494:pharmacology; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D053138:Cyclohexenes; D018744:DNA, Plant / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D011303:Prephenate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology
D000600:Amino Acids, Dicarboxylic; D053138:Cyclohexenes; D018744:DNA, Plant; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D014443:Tyrosine; D009249:NADP; C015478:pretyrosine; D011303:Prephenate Dehydrogenase; C027017:cyclohexadienyl dehydrogenase