Recombinant human tyrosine hydroxylase types 1-4 show regulatory kinetic properties for the natural (6R)-tetrahydrobiopterin cofactor.
Human tyrosine hydroxylase (hTH) exists in four isoforms. The four recombinant hTH isoenzymes types 1-4 (hTH1-4) produced in and purified from Escherichia coli showed regulatory kinetic properties for the natural (6R)-L-erythro-tetrahydrobiopterin (RBPH4) as a cofactor. In contrast, the unnatural cofactor (6S)-L-erythro-tetrahydrobiopterin (SBPH4) and a synthetic cofactor (6RS)-methyl-tetrahydropterin (6MPH4) showed usual kinetic characteristics with each of hTH1-4. Substrate inhibition by tyrosine was observed for each of hTH1-4 with natural RBPH4. Two different Km values for pterin cofactor were observed at a high concentration (200 microM) of L-tyrosine only with natural RBPH4, in contrast to a single Km value for unnatural SBPH4 or synthetic 6MPH4. The present results suggest that in the presence of relatively high concentrations (approximately 100 microM) of tyrosine in vivo, RBPH4 cofactor may have a regulatory role for the activity of all four human isoenzymes in vivo. We also found that recombinant hTH1 and 3 were more unstable than hTH2 and 4, suggesting that the 4-amino-acid insertion in hTH2 and 4 may be responsible for the relative stability of hTH2 and 4 isoenzymes.
Nasrin
S
S
Department of Pharmacology, Nagoya University School of Medicine.
Ichinose
H
H
Hidaka
H
H
Nagatsu
T
T
D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D001708:Biopterins; D014446:Tyrosine 3-Monooxygenase; C003402:sapropterin