Abstract
Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH102-416, was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4α-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 12259-12266 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 273 |
| Issue number | 20 |
| DOIs | |
| State | Published - May 15 1998 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Expression and characterization of the catalytic core of tryptophan hydroxylase. / Moran, Graham R.; Daubner, S. Colette; Fitzpatrick, Paul F.
In: Journal of Biological Chemistry, Vol. 273, No. 20, 15.05.1998, p. 12259-12266.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression and characterization of the catalytic core of tryptophan hydroxylase
AU - Moran, Graham R.
AU - Daubner, S. Colette
AU - Fitzpatrick, Paul F
PY - 1998/5/15
Y1 - 1998/5/15
N2 - Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH102-416, was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4α-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.
AB - Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH102-416, was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4α-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.
UR - http://www.scopus.com/inward/record.url?scp=0032524864&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032524864&partnerID=8YFLogxK
U2 - 10.1074/jbc.273.20.12259
DO - 10.1074/jbc.273.20.12259
M3 - Article
C2 - 9575176
AN - SCOPUS:0032524864
VL - 273
SP - 12259
EP - 12266
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 20
ER -