Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides

Abstract

Inorganic pyrophosphatase containing regulatory cystathionine β-synthase (CBS) domains (CBS-PPase) is inhibited by adenosine monophosphate (AMP) and adenosine diphosphate and activated by adenosine triphosphate (ATP) and diadenosine polyphosphates; mononucleotide binding to CBS domains and substrate binding to catalytic domains are characterized by positive cooperativity. This behavior implies three pathways for regulatory signal transduction — between regulatory and active sites, between two active sites, and between two regulatory sites. Bioinformatics analysis pinpointed six charged or polar amino acid residues of Desulfitobacterium hafniense CBS-PPase as potentially important for enzyme regulation. Twelve mutant enzyme forms were produced, and their kinetics of pyrophosphate hydrolysis was measured in wide concentration ranges of the substrate and various adenine nucleotides. The parameters derived from this analysis included catalytic activity, Michaelis constants for two active sites, AMP-, ATP-, and diadenosine tetraphosphate-binding constants for two regulatory sites, and the degree of activation/inhibition for each nucleotide. Replacements of arginine 295 and asparagine 312 by alanine converted ATP from an activator to an inhibitor and markedly affected practically all the above parameters, indicating involvement of these residues in all the three regulatory signaling pathways. Replacements of asparagine 312 and arginine 334 abolished or reversed kinetic cooperativity in the absence of nucleotides but conferred it in the presence of diadenosine tetraphosphate, without effects on nucleotide-binding parameters. Modeling and molecular dynamics simulations revealed destabilization of the subunit interface as a result of asparagine 312 and arginine 334 replacements by alanine, explaining abolishment of kinetic cooperativity. These findings identify residues 295, 312, and 334 as crucial for CBS-PPase regulation via CBS domains.

LKB1 biology: assessing the therapeutic relevancy of LKB1 inhibitors

“…AMP binding allosterically activates AMPK and protects AMPK from dephosphorylation [ 54 , 55 ]. Ultimately, AMP binding initiates a conformational rearrangement of AMPKɑ in which the catalytic T172 within the T-loop of the kinase domain becomes accessible to upstream AMPK kinases [ 56 ]. Although most investigations of AMPK function and regulation have focused on AMPKɑ and AMPKγ subunits, AMPKβ is necessary for interactions involving carbohydrates [ 57 ], and is post-translationally modified to upregulate or downregulate the enzymatic activity of AMPKɑ [ 58 ].…”

The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine β-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain

“…However, the identity of the structural element that mediates the AMP signal can only be conjectured. Earlier mutagenesis studies of dhPPase have identified two interacting elements, helix α1 of CBS2 domain and the loop formed by residues 310-323 in the DHH domain, among the best candidates for signal transmission from the regulatory site to the catalytic DHH domain [37]. Notably, an Ala substitution for the highly conserved Asn312 in this loop reversed the effect of ATP on dhPPase from activation to inhibition [37].…”

“…Earlier mutagenesis studies of dhPPase have identified two interacting elements, helix α1 of CBS2 domain and the loop formed by residues 310-323 in the DHH domain, among the best candidates for signal transmission from the regulatory site to the catalytic DHH domain [37]. Notably, an Ala substitution for the highly conserved Asn312 in this loop reversed the effect of ATP on dhPPase from activation to inhibition [37]. Furthermore, replacement of Arg168 in the highly conserved RyR motif of Moorella thermoacetica CBS-PPase (corresponding to Arg276 of dhPPase) weakened AMP binding and reversed its effect from inhibition to activation [38].…”

Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase

“…CBS-PPase provides a good model for studying CBS domain-mediated regulation because it is easily accessible and stable, is differentially regulated by adenosine phosphates, including cell alarmones diadenosine polyphosphates, and its activity can be conveniently and precisely measured. In recent site-directed mutagenesis studies of dhPPase, we identified Arg276 and Arg295 in the CBS2 and Asn312 and Arg334 in DHH domains as having crucial roles in kinetic cooperativity (active site interaction) [14,15]. Another important finding was that replacement of Arg295 or Asn312 by alanine reversed the effect of ATP from activation to inhibition [15].…”