TRUNCATION AND ALANINE-SCANNING MUTANTS OF TYPE-I ADENYLYL-CYCLASE

A variety of truncated constructs of type I and type II adenylyl cycla se have been synthesized in Sf9 cells using recombinant baculoviruses, as have a number of type I adenylyl cyclases with point mutations. Tr uncations indicate that the nonconserved C-1b and C-2b domains of aden ylyl cyclase are not necessary for regulation of enzymatic activity by G(s alpha) and forskolin. Point mutations demonstrate the requirement for both of the conserved (and homologous) domains of adenylyl cyclas e (C-1a and C-2a) and the nonequivalence of these domains. Linkage of certain effects of mutations on the K-m for substrate with alterations of the characteristics of beta-site inhibition suggest that ATP and b eta-site inhibitors may bind to different conformations of the same si te. However, other mutations affected only alpha-site inhibition. Alth ough the mutations studied have not permitted assignment of unique fun ctions to the two homologous domains, they have revealed novel phenoty pes that appear to reflect the regulatory complexity of mammalian memb rane-bound adenylyl cyclases, including the possibility of oligomeriza tion of the enzymes.