Kinetic properties of 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase from Escherichia coli.
Kinetic properties of purified 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMSA) dehydrogenase (EC 1.2.1.-) in the 4-hydroxyphenylacetate meta-cleavage pathway from Escherichia coli have been studied. The temperature--activity relationship for the enzyme from 27 to 45 degrees C showed an Arrhenius plot with an inflexion at 36 degrees C. When 5-carboxymethyl-2-hydroxymuconic semialdehyde and NAD were used as variable substrates, the double reciprocal plots were all linear and the lines intersected at one point below the horizontal axis, suggesting that a sequential mechanism is operating. From the replots of intercepts and slopes against reciprocal substrate concentrations were calculated Km (CHMSA) = 9.0 +/- 1.02 microM, Km (NAD) = 29.1 +/- 4.65 microM and the value for the dissociation constant of enzyme--NAD complex = 6.3 +/- 1.21 microM. ATP and the product of the reaction (NADH) acted as competitive inhibitors of the enzyme with respect to NAD. Apparent Ki values, estimated from Dixon plots, were 25.0 +/- 3.5 and 88.0 +/- 22.1 microM for NADH and ATP, respectively.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature
D000447:Aldehydes; D009243:NAD; C015930:2-hydroxy-5-carboxymethylmuconate semialdehyde; D000255:Adenosine Triphosphate; D000445:Aldehyde Oxidoreductases; C026251:5-carboxymethyl-2-hydroxymuconic acid semialdehyde dehydrogenase