On the catalytic mechanism of tryptophan hydroxylase

Tryptophan hydroxylase catalyzes the hydroxylation of tryptophan using tetr ahydrobiopterin and molecular oxygen. With tyrosine as a substrate, the amo unt of C4a-hydroxypterin formed greatly exceeds the amount of dihydroxyphen ylalanine formed, consistent with oxygen-oxygen bond cleavage occurring in a step prior to amino acid hydroxylation. With L-indole-H-2(5)-tryptophan, L-4-H-2- Or L-5-H-2-tryptophan as substrate there is no isotope effect on t he V/K value for tryptophan. There is an inverse isotope effect on the V-ma x value with L-indole-B-2(5)-tryptophan and L-5-H-2-tryptophan, but no effe ct with L-4-H-2-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 format ion of a cationic species. Retention of the isotopic label from L-5-H-2-try ptophan in the product confirms that an NIH shift occurs in tryptophan hydr oxylase 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 i nitially on carbon 4 rather than carbon 5.