PubMed · 3565767
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M Y Ho, G A Rechnitz. 1987-02-01. Highly stable biosensor using an artificial enzyme.. https://doi.org/10.1021/ac00130a038
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Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.
Oxaloacetate decarboxylase of Klebsiella pneumoniae was shown to contain between 0.6 and 1.0 mol zinc per mol enzyme in different preparations. The decarboxylase activity was completely abolished after 15 min incubation with 1 mM Hg(NO3)2 in phosphate buffer, while the activity decreased only 20% if the incubation was performed in MES/Tris buffer. Treatment of the isolated subunits with Hg(NO3)2 indicated that the binding site for Hg2+ ions is on the alpha subunit. Other inhibitors of the decarboxylase are KSCN and diethylstilbestrol. Inactivation of the enzyme with 2% 1-butanol was significantly reduced by 100 mM NaCl. Sodium ions also protected the isolated beta + gamma subunits from a digestion with trypsin.
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