Influence of steric bulk and electrostatics on the hydroxylation regiospecificity of tryptophan hydroxylase: Characterization of methyltryptophans and azatryptophans as substrates

Graham R. Moran, Robert S. Phillips, Paul F. Fitzpatrick

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

Tryptophan hydroxylase is a pterin-dependent amino acid hydroxylase that catalyzes the incorporation of one atom of molecular oxygen into tryptophan to form 5-hydroxytryptophan. The substrate specificity and hydroxylation regiospecificity of tryptophan hydroxylase have been investigated using tryptophan analogues that have methyl substituents or nitrogens incorporated into the indole ring. The products of the reactions show that the regiospecificity of tryptophan hydroxylase is stringent. Hydroxylation does not occur at the 4 or 6 carbon in response to changes in substrate topology or atomic charge. 5-Hydroxymethyltryptophan and 5-hydroxy-4-methyltryptophan are the products from 5-methyltryptophan. These products establish that the hydroxylating intermediate is sufficiently potent to hydroxylate benzylic carbons and that the direction of the NIH shift in tryptophan hydroxylase is from carbon 5 to carbon 4. The effects on the V/K values for the amino acids indicate that the enzyme is most sensitive to changes at position 5 of the indole ring. The V(max) values for amino acid hydroxylation differ at most by a factor of 3 from that observed for tryptophan, while the efficiencies of hydroxylation with respect to tetrahydropterin consumption vary 6-fold, consistent with oxygen transfer to the amino acid being partially or fully rate limiting in productive catalysis.

Original languageEnglish (US)
Pages (from-to)16283-16289
Number of pages7
JournalBiochemistry
Volume38
Issue number49
DOIs
StatePublished - Dec 7 1999

ASJC Scopus subject areas

  • Biochemistry

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