MUTATIONAL INACTIVATION OF THE CATALYTIC DOMAIN OF GUANYLATE CYCLASE-A RECEPTOR

Guanylate cyclase-A, the receptor for atrial natriuretic factor, conta ins a protein kinase-like domain and a catalytic domain in the intrace llular region. To investigate the active site (the catalytic cavity) o f guanylate cyclase-A, we amplified the catalytic domain plus three am ino acids from the kinase-like domain of guanylate cyclase-A (GC-c) wi th polymerase chain reaction (PCR) and expressed it in Escherichia col i. During the screening of the PCR-cloned gene products with guanylate cyclase assay, a mutant that lacks enzyme activity was identified. Re sults of cDNA sequencing revealed that Leu 817 was replaced by an Arg residue in the mutated protein. The mutated GC-c bound to GTP-agarose as well as the wild-type protein, indicating that the binding capabili ty of mutated GC-c to GTP is not significantly affected by the Arg sub stitution. Gel-filtration column chromatography showed that, like the wild-type GC-c, the mutated protein also formed a high-molecular-weigh t complex. Since mutation of Leu 817 to Arg abolishes the catalytic ac tivity, Leu 817 is likely located near the active site of guanylate cy clase-A. These results demonstrate that the carboxyl fragment of guany late cyclase-A is an ideal system for studying the active site of guan ylate cyclase-A.