Functional Characterization of Two Nucleotide-binding Sites in Soluble Guanylate Cyclase

Shinsuke Yazawa,Hidemi Tsuchiya,Hiroshi Hori,Ryu Makino

doi:10.1074/jbc.m508983200

Shinsuke Yazawa, Hidemi Tsuchiya + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m508983200

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Abstract

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of alpha- and beta-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for alpha,beta-methylene guanosine 5'-triphosphate (GMP-CPP). In contrast, 2'-dADP occupied both sites with equivalent affinities. Adenosine-5'-beta,gamma-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2'-dADP, probably by competing for the same site as 2'-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2'-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.

Highlights

Heme via a weak His-iron bond [11,12,13]
The Kd of 38.4 ␮M in the presence of Mn2ϩ at 25 °C essentially agreed with Ki determined kinetically under comparable conditions (ϳ30 ␮M at 27 °C), as shown in the inset of Fig. 3B. These findings indicate that 2Ј-dADP binds to the high and low affinity sites with equivalent affinities and suggest that the noncompetitive inhibitory site corresponds to the low affinity site
The addition of YC-1 fully converted the AMP-PNPbound NO enzyme to a NO-heme species with a rhombic EPR signal (Fig. 8, compare trace d with trace e). These results imply that YC-1 barely associates with the 2Ј-dADP-enzyme complex but readily binds to the AMP-PNP-enzyme complex even in the current biochemical investigations of Soluble guanylate cyclase (sGC) provide precise information concerning the activation of the NO receptor by NO, most of these studies have focused on the reaction of the heme group with NO [12, 13, 17, 18, 40]

Summary

Introduction

Heme via a weak His-iron bond [11,12,13]. The binding of NO to the heme markedly stimulates the enzymatic production of cGMP (9, 14 –16). A Scatchard plot for the binding of Mn2ϩ-GMP-CPP becomes curved at elevated concentrations of the nucleotide, representing the presence of the high and low affinity sites for the binding of this nucleotide (Fig. 1, inset). We propose that the enzyme has both high and low affinity binding sites for the nucleotide.

Results

Conclusion