On the Catalytic Mechanism of Tryptophan Hydroxylase

Abstract

Tryptophan hydroxylase catalyzes the hydroxylation of tryptophan using tetrahydrobiopterin and molecular oxygen. With tyrosine as a substrate, the amount of C4a-hydroxypterin formed greatly exceeds the amount of dihydroxyphenylalanine formed, consistent with oxygen−oxygen bond cleavage occurring in a step prior to amino acid hydroxylation. With l-indole-2H5-tryptophan, l-4-2H- or l-5-2H-tryptophan as substrate there is no isotope effect on the V/K value for tryptophan. There is an inverse isotope effect on the Vmax value with l-indole-2H5-tryptophan and l-5-2H-tryptophan, but no effect with l-4-2H-tryptophan. Comparison of the measured isotope effects with values of calculated secondary equilibrium isotope effects for tryptophan hydroxylation indicate that the results are most consistent with the formation of a cationic species. Retention of the isotopic label from l-5-2H-tryptophan in the product confirms that an NIH shift occurs in tryptophan hydroxylase and shows that the direction of shift is from carbon 5 to carbon 4. The degree of retention of the deuterium is higher when the deuterium is initially on carbon 4 rather than carbon 5.