Substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli.
The investigation of the substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli allows a description of the binding region of pyruvate. Substrate analogs with electronegative substitutions in the methyl group show a strong competitive inhibition of the overall reaction of the pyruvate dehydrogenase complex. The most efficient inhibitor is fluoropyruvate which has a more than 100-fold higher affinity for the enzyme than pyruvate (Ki = 1.4 x 10(-6) M) does. The affinity of alpha-keto acids decreases with increasing chain length. Branched chain alpha-keto acids are even less effective inhibitors (Ki = approximately 0.02 M). alpha-Ketobutyrate is the only alpha-keto acid which is able to substitute for pyruvate as a substrate in the overall reaction of the enzyme complex. The Km value (3 mM) is 10-fold greater than that for pyruvate. The steady state kinetics of the overall reaction of alpha-ketobutyrate exhibits the same cooperativity (nh = 1.9) as seen with pyruvate. Small modifications of the carbonyl or the carboxyl group of pyruvate prevent binding completely. Binding of pyruvate to the pyruvate dehydrogenase complex may thus require interaction with two independent electrophilic centers. The acceptance of the methyl group seems not so much due to lipophilic interactions as to a steric effect. The experiments were carried out with an enzyme which was purified by a modified procedure which is faster and more convenient than previous methods. The procedure is applicable up to 0.5 liter of crude extract.
D001665:Binding Sites; D002264:Carboxylic Acids / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D011485:Protein Binding; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D013379:Substrate Specificity
D002264:Carboxylic Acids; D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; C017092:bromopyruvate; C012375:hydroxypyruvic acid