2 AMINO-ACID SUBSTITUTIONS CONVERT A GUANYLYL CYCLASE, RETGC-1, INTO AN ADENYLYL-CYCLASE

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental r oles in a wide range of cellular processes. Whereas GCs use GTP as a s ubstrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP, Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replace d specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity f rom GTP to ATP. These experiments strongly validate the AC-derived Ret GC-1 structural model and functionally confirm the role of these resid ues in nucleotide discrimination.