Guanylyl Cyclases A and B Are Asymmetric Dimers That Are Allosterically Activated by ATP Binding to the Catalytic Domain

Abstract

It is not known how natriuretic peptides and adenosine triphosphate (ATP) activate guanylyl cyclase A (GC-A) and GC-B, which generate the second messenger cyclic guanosine monophosphate. We determined that natriuretic peptides increased the maximum rate of these enzymes >10-fold in a positive cooperative manner in the absence of ATP. In the absence of natriuretic peptides, ATP shifted substrate-velocity profiles from cooperative to linear but did not increase the affinity of GCs for the substrate guanosine triphosphate (GTP) since the Michaelis constant was unchanged. However, in the presence of natriuretic peptides, ATP competed with GTP for binding to an allosteric site, which enhanced the activation of GCs by decreasing the Michaelis constant. Thus, natriuretic peptide binding was required for communication of the allosteric activation signal to the catalytic site. The ability of ATP to activate GCs decreased and enzyme potency (a measure of sensitivity to stimulation) increased with increasing GTP concentrations. Point mutations in the purine-binding site of the catalytic domain abolished GC activity but not allosteric activation. Coexpression of inactive mutants produced half the activity expectedfor symmetric catalytic dimers. 2′-Deoxy-ATP and 2′-deoxy-GTP were poorallosteric activators, but 2′-deoxy-GTP was an effective substrate, consistent with distinct binding requirements for the allosteric and catalytic sites. We conclude that membrane GC domains are asymmetric homodimers with distinct and reciprocally regulated catalytic and allosteric sites that bind to GTP and ATP, respectively. These data define a new membrane GC activation model and provide evidence of a previously unidentified GC drug interaction site.

A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B

“…Basal activity of the WT enzyme was low and CNP increased Vmax 12-fold without decreasing the Km. Consistent with previous observations [6], WT-GC-B was positive cooperative as indicated by a Hill slope of 1.3. In contrast, basal maximal velocity of the mutant enzyme was elevated 15-fold compared to WT-GC-B and the Km was unchanged.…”

“…2). Enzyme analysis was performed in the 293 cells because they do not express detectable endogenous GC activity [6], which allows more definitive interpretation of the data because most tissues and cell lines express more than one GC.…”

“…However, under biological conditions where ATP concentrations are at or above 1 mM, the Hill coefficient of GC-B is 1 because the allosteric site is occupied by ATP not GTP. Recently, we demonstrated that ATP is required for the CNP-dependent reduction in the Km of GC-B [5,6]. Finally, in broken cell assays, ATP also increases GC-B activity by providing the phosphate that is added to the serine and threonine residues on the enzyme that is necessary for activation by CNP [7,8].…”

Structure/Activity Relationships of (M)ANT- and TNP-Nucleotides for Inhibition of Rat Soluble Guanylyl Cyclase α1β1

“…However, such studies are worthwhile, and we expect a different inhibitor profile for pGC compared with sGC and mACs. First steps toward the development of selective allosteric pGC modulators have already been taken (Robinson and Potter, 2012;Seifert and Beste, 2012). Identification of high-potency pGC-A inhibitors may support biophysical studies with this enzyme.…”

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism

“…GnT1 293 cells lacking the Golgi N-acetylglucosaminyltransferase I enzyme (29) were purchased from the ATCC (Manassas, VA). 293T cells were transfected using the calcium/phosphate method as described previously (30). For HeLa cells, a 1:2 mass ratio of plasmid DNA to Lipofectimine2000 was incubated in 0.5 ml of Opti-MEM for 20 min before addition to cells.…”