On the Kinetics and Mechanism of Enoate Reductase

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Abstract
Enoate reductases from Clostridium spec. La 1 and Clostridium kluyveri show a rather broad substrate specificity i.e. many alpha,beta-unsaturated carboxylates are reduced in a NADH-dependent reaction. The relative rates for different substrates are different for both reductases. The Km value of NADH for the reductase from C. spec. La 1 is about 12 muM. The transhydrogenase activity (reduction of N-acetylpyridine adenine dinucleotide) with NADH shows a maximum at pH 8 which is about 2 units higher than that for the reduction of enoates. Results of initial rate studies can be best explained by a Bi Bi ping pong mechanism. No back reaction and no proton exchange from 2(-3)H-labelled acylates could be demonstrated. NAD+ is a mixed-type inhibitor. The product inhibition constant Ki = 0.84mM and the dissociation constant for the dead-end inhibition complex 4.8mM. Aliphatic acylates show no measurable inhibition when they are applied in concentrations at the 100-fold Km values of the corresponding enoates. Measurable inhibitions can be observed with phenyl group-containing acylates. 3-Phenylpropionate (38mM) shows about 86% inhibition. Fumarate which is not a substrate inhibits the reduction of enoates by NADH as well as by reduced methylviologen. However, the reduction of NAD+ by reduced methylviologen as well that of acetylpyridine adenine dinucleotide by NADH is not inhibited by fumarate. On the other hand inhibitors such as morin or dicoumarol which probably bind to the flavin domain do not impair the reduction of enoates by reduced methylviologen however, all reductions with NADH are inhibited. These results are indicative for three binding domains: one for NADH which can be blocked by dicoumarol or morin, another for enoates which can be occupied by fumarate and a third one for reduced methylviologen. Enoate reductase splits off exclusively the (4S)-hydrogen atom from NADH. There is no direct hydrogen transfer from NADH to the products. Depending on the substrate concentration the isotope effect of the reduction of (E)-2-methyl-2-butenoate with (4S)-[4(-3)H]NADH varies from 6.8 to 1.3. The presence of NAD+ decreases the isotope effect.