Expression and characterization of recombinant human and rat liver 6-pyruvoyl tetrahydropterin synthase. Modified cysteine residues inhibit the enzyme activity.
6-Pyruvoyl-tetrahydropterin synthase is the rate-limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. The human and rat liver cDNAs encoding the 16-kDa subunit of 6-pyruvoyl tetrahydropterin synthase were expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion proteins. After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity. Apparent Km for the substrate dihydroneopterin triphosphate (8.5 microM for the human and 8.0 microM for the rat enzyme), pI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes. The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification. The modification of the only cysteine residue in rat 6-pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80%. Modification under non-reducing conditions of both cysteine residues of the human enzyme by N-ethylpyridine resulted in a 95% loss of enzyme activity. This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis. Cross-linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits.
Bürgisser
D M
DM
Department of Pediatrics, University of Zürich, Switzerland.
Thöny
B
B
Redweik
U
U
Hunziker
P
P
Heizmann
C W
CW
Blau
N
N
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D004926:Escherichia coli; D005033:Ethylmaleimide / Q000494:pharmacology; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019761:Phosphorus-Oxygen Lyases; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D017931:DNA Primers; D006146:Guanidines; D011725:Pyridines; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D015951:Factor Xa; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; C029351:4-vinylpyridine; D019791:Guanidine; D003545:Cysteine; D005033:Ethylmaleimide