IMP dehydrogenase inhibitors reduce intracellular tetrahydrobiopterin levels through reduction of intracellular GTP levels. Indications of the regulation of GTP cyclohydrolase I activity by restriction of GTP availability in the cells.

K Hatakeyama,T Harada,H Kagamiyama

doi:10.1016/s0021-9258(19)36747-x

K Hatakeyama, T Harada + Show 1 more

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https://doi.org/10.1016/s0021-9258(19)36747-x

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Abstract

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.

Highlights

INDICATIONSOF THE REGULATION OF GTP CYCLOHYDROLASE I ACTIVITY BY RESTRICTION OF GTP AVAILABILITY IN THE CELLS*
The amount of biopterin released into the me- lase I is the rate-limiting step in the biosynthesis of BH, in dium was decreased concomitantly with the decrease in PC-12 cells (Table 11), the similarity between the substrateintracellular BH,. These results suggested that the biopterin velocity curve of GTP cyclohydrolase I (Fig. 5 A ) and the production in PC-12 cells declined as a resultof the treatment GTP-biopterin curves (Fig. 3) suggests that the enzyme with IMP dehydrogenase inhibitors
I M P Dehydrogenase Inhibitors Reduce Intracellular Tetrahydrobiopterin Levelsvia Reduction of Intracellular GTP Lev&-In the present study, we have demonstrated that IMP dehydrogenase inhibitors lower the levels of intracellular tetrahydrobiopterin in PC-12 rat pheochromocytoma and IMR32 human neuroblastoma cells

Summary

RESULTS

Dehydrogenase inhibitors and/or guanine compounds as indicated in I M P DehydrogenaseInhibitors Decrease GTP and Biopterin the text. The amount of pterin in the medium was decreased with between GTP andbiopterin levels, we examined the effect of increasing concentrations of the inhibitors, concurrent with mycophenolic acid on IMR-32 cells that were derived from the reduction in biopterin in the cells. Guanosine or guanine added simultaneously with the inhibitors completely reversed the decrease in biopterin level caused by the inhibitors This result suggests that there is a close cellular concentrations of GTP were found in the range effectively altering the biopterin level in neuronal cell lines from two different species. Enzymes were extracted and assayed as described under “Experimental Procedures.” Values shown represent means f S.E. from two identical experiments performed in separate cultures

DISCUSSION

Findings

Regulation of GTP Cyclohydrolase I by GTP Levels