Metabolism of aromatic compounds by fungi. Kinetic properties and mechanism of 3-carboxy-cis,cis-muconate cyclase from Aspergillus niger.
A preliminary investigation of the kinetic properties of 3-carboxy-cis,cis-muconate cyclase (EC 5.5.1.5) has been performed. The initial velocity of the reaction was shown to be proportional to the concentration of the enzyme in the assay system adopted and the apparent Km was found to be 57 muM at pH 6.0 and 30 degrees C but at concentrations exceeding 70 muM, substrate inhibition was apparent. At pH 6.0 the Ki for the substrate was 0.45 mM. Plots of V and Km against pH showed inflexions at pH 5.3 and pH 6.4. The enzyme was inhibited by a variety of inorganic anions and by a number of dicarboxylic and tricarboxylic acids. The degree of inhibition exerted by these acids was found to be proportional to the proximity of their carboxyl groups, the cis configuration being a more effective inhibitor than the trans configuration. As inhibition was competitive in each case, the presence of an anion-sensitive substrate-binding site has been postulated. The cis-cis, cis-trans and trans-trans isomers of muconate, 3-chloromuconate and 3-carboxy-cis-trans-muconate, close analogues of natural substrate but not attacked by the enzyme, were also found to be competitive inhibitors. The variation in pKi with pH was determined in the case of cis,cis-muconate and cis-aconitate, both of which gave curves suggesting the importance of a group with a pKa of approximately 6.4 responsible for increasing the inhibition of the enzyme. Modification by ethoxyformic anhydride and the kinetics of Rose-Bengal-sensitized photo-oxidation suggested the participation of a histidine residue in the catalytic reaction. These results are discussed in the light of recent work on enzymes catalysing analogous reactions; a likely reaction mechanism has been proposed.
D001230:Aspergillus / Q000201:enzymology*; D001234:Aspergillus niger / Q000201:enzymology*; D002264:Carboxylic Acids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019753:Intramolecular Lyases; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D007783:Lactones; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D010777:Photochemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D002264:Carboxylic Acids; D007783:Lactones; D007535:Isomerases; D019753:Intramolecular Lyases; C019831:3-carboxy-cis-cis-muconate cyclase