Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa.
Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000596:Amino Acids / Q000302:isolation & purification; D000603:Amino Acids, Sulfur / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D008974:Mollusca / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology; D013379:Substrate Specificity
D000596:Amino Acids; D000603:Amino Acids, Sulfur; D007527:Isoenzymes; C050557:tauropine; D000594:Amino Acid Oxidoreductases; C050564:tauropine dehydrogenase