Mutational inactivation of the catalytic domain of guanylate cyclase-A receptor.
Guanylate cyclase-A, the receptor for atrial natriuretic factor, contains a protein kinase-like domain and a catalytic domain in the intracellular region. To investigate the active site (the catalytic cavity) of guanylate cyclase-A, we amplified the catalytic domain plus three amino acids from the kinase-like domain of guanylate cyclase-A (GC-c) with polymerase chain reaction (PCR) and expressed it in Escherichia coli. During the screening of the PCR-cloned gene products with guanylate cyclase assay, a mutant that lacks enzyme activity was identified. Results 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 capability of mutated GC-c to GTP is not significantly affected by the Arg substitution. Gel-filtration column chromatography showed that, like the wild-type GC-c, the mutated protein also formed a high-molecular-weight complex. Since mutation of Leu 817 to Arg abolishes the catalytic activity, Leu 817 is likely located near the active site of guanylate cyclase-A. These results demonstrate that the carboxyl fragment of guanylate cyclase-A is an ideal system for studying the active site of guanylate cyclase-A.
Miao
Z H
ZH
Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Song
D L
DL
Douglas
J G
JG
Chang
C H
CH
D000262:Adenylyl Cyclases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism
D015345:Oligonucleotide Probes; C068914:guanosine triphosphate-agarose; D006160:Guanosine Triphosphate; D012685:Sepharose; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor