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 hetero logous gene expression in Escherichia coli and characterized by bioche mical and biophysical methods. Correlation averaged electron micrograp h images of preferentially oriented enzyme particles revealed a fivefo ld rotational symmetry of the doughnut-shaped views with an average pa rticle diameter of 10 nm. Analytical ultracentrifugation and quantitat ive scanning transmission electron microscopy yielded average molecula r masses of 270kDa and 275kDa, respectively. Like the Escherichia coli homolog, these findings suggest that the active enzyme forms a homode cameric protein complex consisting of two fivefold symmetric pentameri c rings associated face-to-face. Examination of the amino acid sequenc e combined with calcium-binding experiments and mutational analysis re vealed a high-affinity, EF-hand-like calcium-binding loop motif in euk aryotic enzyme species, which is absent in bacteria. Intrinsic fluores cence 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 design ed mutations within the presumed calcium-binding loop of the rat GTP c yclohydrolase I yielded a 45% decrease in enzyme activity. This findin g suggests that failure of calcium binding may be the consequence of a mutation recently identified in the causative GTP cyclohydrolase I ge ne of patients suffering from dopa responsive dystonia. (C) 1998 Acade mic Press Limited.