Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2.
Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.
Winge
Ingeborg
I
Department of Biomedicine, Section of Biochemistry and Molecular Biology, University of Bergen, Norway.
McKinney
Jeffrey A
JA
Knappskog
Per M
PM
Haavik
Jan
J
D002461:Cell Line, Transformed; D004298:Dopamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D014364:Tryptophan / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000502:physiology*
D014364:Tryptophan; C494784:TPH2 protein, human; D014365:Tryptophan Hydroxylase; D004298:Dopamine