Functional Characterization of Nitric Oxide and YC-1 Activation of Soluble Guanylyl Cyclase: Structural Implication for the YC-1 Binding Site?

Maria Lamothe, Fu Jung Chang, Nataliya Balashova, Roman Shirokov, Annie Beuve

Research output: Contribution to journalArticlepeer-review

81 Scopus citations

Abstract

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an α subunit and a β subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.

Original languageEnglish (US)
Pages (from-to)3039-3048
Number of pages10
JournalBiochemistry
Volume43
Issue number11
DOIs
StatePublished - Mar 23 2004

All Science Journal Classification (ASJC) codes

  • Biochemistry

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