Use of alternate substrates to probe the order of substrate addition to dopamine β-hydroxylase

In order to determine the order of substrate binding to dopamine β-hydroxylase during catalysis, the effect of alternate substrates upon kinetic parameters was examined. The V K value for ascorbate was unchanged when tyramine, phenylpropylamine, p-Cl-phenethylamine, p-CH₃O-phenethylamine, or phenethylamine was the hydroxylated substrate. The V K values for tyramine and oxygen were similarly unchanged when ferrocyanide was used as the reductant in place of ascorbate. In order to use ferrocyanide as reductant it was necessary to include copper to alleviate the substrate inhibition seen with this substrate. The pattern of substrate inhibition observed with ferrocyanide was consistent with a small amount of free cyanide present in the ferrocyanide. With ferrocyanide as reductant and [2,2-²H₂]tyramine as substrate, there was a measurable isotope effect on the V K value for oxygen, but none on the values of V_max or V K for tyramine. These results are consistent with a ping-pong mechanism in which tyramine binds to the enzyme after the release of oxidized ascorbate. Subsequently, oxygen binds to form a ternary complex.