On the catalytic mechanism of tryptophan hydroxylase

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 V(max) 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.