The presence of essential histidine residues in manganese(III)-containing acid phosphatase from sweet potato.
Chemical modification studies of manganese(III)-containing acid phosphatase [EC 3.1.3.2] were carried out to investigate the contributions of specific amino-acid side-chains to the catalytic activity. Incubation of the enzyme with N-ethylmaleimide at pH 7.0 caused a significant loss of the enzyme activity. The inactivation followed pseudo-first-order kinetics. Double log plots of pseudo-first-order rate constant vs. concentration gave a straight line with a slope of 1.02, suggesting that the reaction of one molecule of reagent per active site is associated with activity loss. The enzyme was protected from inactivation by the presence of molybdate or phosphate ions. Amino acid analyses of the N-ethylmaleimide-modified enzyme showed that the 96%-inactivated enzyme had lost about one histidine and one-half lysine residue per enzyme subunit without any significant decrease in other amino acids, and also demonstrated that loss of catalytic activity occurred in parallel with the loss of histidine residue rather than that of lysine residue. Molybdate ions also protected the enzyme against modification of the histidine residue. The enzyme was inactivated by photooxidation mediated by methylene blue according to pseudo-first-order kinetics. The pH profile of the inactivation rates of the enzyme showed that an amino acid residue having a pKa value of approximately 7.2 was involved in the inactivation. These studies indicate that at least one histidine residue per enzyme subunit participates in the catalytic function of Mn(III)-acid phosphatase.
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000032:analysis; D008345:Manganese / Q000032:analysis*; D010084:Oxidation-Reduction; D010777:Photochemistry; D010944:Plants / Q000201:enzymology*
D000596:Amino Acids; D008345:Manganese; D006639:Histidine; D000135:Acid Phosphatase; D008239:Lysine; D005033:Ethylmaleimide