The Interaction of GTP Cyclohydrolase I and GTP Cyclohydrolase Feedback Regulatory Protein Can be Detected using the Yeast Two-Hybrid System
The enzyme GTP cyclohydrolase I (GTPCH) catalyzes the first and rate-limiting step in the synthesis of tetrahydrobiopterin (BH4) (1), the required cofactor for tyrosine and tryptophan hydroxylases (2). All members of the family of nitric oxide synthases also require BH4 (3). Intracellular concentrations of BH4 are normally subsaturating for these enzymes (4) and alterations in BH4 levels brought about by changes in GTPCH activity can modify the synthesis of monoamine and nitric oxide neurotransmitters. GTPCH has a regulatory subunit known as GTP cyclohydrolase feedback regulatory protein (GFRP) (5). In the presence of GFRP BH4 inhibits GTPCH activity thereby inhibiting BH4 synthesis. The interaction between GTPCH and GFRP is not completely understood, but it is thought that two pentameric molecules of GFRP bind to the outer surface of the GTPCH homodecamer (6). We have begun to use the yeast two-hybrid system to study the protein domains required for the association of GTPCH and GFRP. Our results indicate that the two-hybrid system can detect a strong interaction between GTPCH and GFRP. More detailed analysis shows that deletion of the N-terminal domain of GTPCH eliminates the interaction between GTPCH and GFRP. We propose that the association of GFRP and GTPCH involves the N-terminal, proline-rich domain of GTPCH and possibly SH3-like regions in GFRP.
KeywordsZinc Agar Lithium DMSO Tyrosine
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