A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization |
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Authors: | Nielsen Michael S Petersen Charlotte R Munch Astrid Vendelboe Trine V Boesen Jane Harris Pernille Christensen Hans E M |
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Institution: | Department of Chemistry, Building 207, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark. |
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Abstract: | Tryptophan hydroxylase (TPH) EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected. |
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Keywords: | Tryptophan hydroxylase Expression Purification Catalytic domain Kinetic Crystallization |
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