pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications.
Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine. The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag. When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine. Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8. These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8. Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4-dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.
D000494:Allosteric Regulation; D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008546:Melanoma, Experimental / Q000201:enzymology*; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012995:Solubility; D014443:Tyrosine / Q000494:pharmacology; D014867:Water
D014867:Water; D014443:Tyrosine; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase