Purification and characterization of polyphenol oxidase from potato: II. Inhibition and catalytic mechanism

KCN and ascorbic acid showed competitive inhibition patterns with K-is values of 0.032 and 0.27 mM, respectively. Uncompetitive inhibition patterns were obtained with sodium azide, L-cysteine and NaCl with K-ii values of 3.3 mM, 0.12 mM and 0.3 M, respectively. A noncompetitive inhibition pattern was obtained for thiourea with 0.067 mM for K-is and 0.59 mM for K-ii. Cu2+ increased the activity about 2.5 fold at or above 40 mu M and K+ decreased the enzyme activity about 33% at 0.4 M. Other metal ions did not have any effects on the activity. Two pK values of 5.8 and 8.0 were obtained from V-max profile and two pK values of 5.9 and 8.1 from V-max/K-m profile. The data suggest that cysteine is likely to be involved in catalysis and histidine in binding. Data from chemical modification show that cysteine was completely inactivated at 1.74 mM o-methylisourea, and histidine and tryptophan were modified at much higher concentrations of diethylpyrocarbonate and N-bromosuccinimide, respectively. It is suggested that the protonated cysteine works as a general base, tryptophan as a substrate binding residue and histidine as a oxygen binding residue.