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J. Biol. Chem., Vol. 259, Issue 3, 1601-1606, 02, 1984

Alternate substrates of dopamine beta-hydroxylase. II. Inhibition by benzyl cyanides and reactivation of inhibited enzyme

G Colombo, DP Giedroc, B Rajashekhar and JJ Villafranca

Several ring-substituted benzyl cyanides lead to inactivation of dopamine beta-hydroxylase during catalysis. With m-hydroxybenzyl cyanide, maximal inactivation occurs when an enzyme group with a pK alpha of 6.0 +/- 0.2 is ionized (Colombo, G., Rajashekhar, B., Giedroc, D. P., and Villafranca, J.J. (1984) J. Biol. Chem. 259, 1593-1600). This paper reports studies conducted to determine the stability of inactivated dopamine beta-hydroxylase. Inactivation of the enzyme by m- hydroxybenzyl cyanide at pH 6.4 is halted by lowering the pH to approximately 5.0 with acetate, fumarate, pyridine, or phosphate buffer in the presence of tyramine. However, if tyramine is omitted, reactivation occurs. The extent of reactivation is dependent upon the final pH value and buffer used to adjust the pH. Reactivation is observed as the pH is lowered from 6.4 to below 5.7 with acetate, fumarate, or HCl. With phosphate, reactivation occurs at any pH value from 6.9 to 4.5 but is greater at lower pH values. Thus, inactivation and reactivation have opposite pH dependencies. Also, reactivation is dependent upon the elapsed time of inactivation. At early times, no reactivation is observed when phosphate is used to adjust the pH, but reactivation is observed later in the inactivation reaction. Reactivation to 100% of the original activity does not occur under these conditions. These data suggest at least two inactivation mechanisms by benzyl cyanides: 1) formation of a tightly bound or covalent adduct between dopamine beta-hydroxylase and enzyme-bound mandelonitrile (or a rearranged form of this molecule), and 2) reversible inhibition resulting from cyanide binding to enzyme-Cu2+. Studies with radiolabeled p-hydroxybenzyl cyanide as well as EPR studies of dopamine beta-hydroxylase-Cu2+ are reported in the following paper.
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