Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites.
Recombinant rat liver GTP cyclohydrolase I has been prepared by heterologous gene expression in Escherichia coli and characterized by biochemical and biophysical methods. Correlation averaged electron micrograph images of preferentially oriented enzyme particles revealed a fivefold rotational symmetry of the doughnut-shaped views with an average particle diameter of 10 nm. Analytical ultracentrifugation and quantitative scanning transmission electron microscopy yielded average molecular masses of 270 kDa and 275 kDa, respectively. Like the Escherichia coli homolog, these findings suggest that the active enzyme forms a homodecameric protein complex consisting of two fivefold symmetric pentameric rings associated face-to-face. Examination of the amino acid sequence combined with calcium-binding experiments and mutational analysis revealed a high-affinity, EF-hand-like calcium-binding loop motif in eukaryotic enzyme species, which is absent in bacteria. Intrinsic fluorescence measurements yielded an approximate dissociation constant of 10 nM for calcium and no significant binding of magnesium. Interestingly, a loss of calcium-binding capacity observed for two rationally designed mutations within the presumed calcium-binding loop of the rat GTP cyclohydrolase I yielded a 45% decrease in enzyme activity. This finding suggests that failure of calcium binding may be the consequence of a mutation recently identified in the causative GTP cyclohydrolase I gene of patients suffering from dopa responsive dystonia.
Steinmetz
M O
MO
Maurice E. Müller Institute for Microscopy, Biozentrum University of Basel, Switzerland.
Plüss
C
C
Christen
U
U
Wolpensinger
B
B
Lustig
A
A
Werner
E R
ER
Wachter
H
H
Engel
A
A
Aebi
U
U
Pfeilschifter
J
J
Kammerer
R A
RA
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D006136:GTP Cyclohydrolase / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D005818:Genetic Engineering; D008099:Liver / Q000201:enzymology; D008854:Microscopy, Electron; D011487:Protein Conformation; D051381:Rats
D006136:GTP Cyclohydrolase; D002118:Calcium