Roles of nucleotide substructures in the regulation of cystathionine β-synthase domain-containing pyrophosphatase

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 8; pp. 1263 - 1269
• Main Authors: Anashkin, Viktor A., Aksenova, Vera A., Vorobyeva, Natalya N., Baykov, Alexander A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2019
• Subjects: active sites; Adenine; Adenine Nucleotides - chemistry; Adenosine; adenosine diphosphate; adenosine monophosphate; adenosine triphosphate; Allosteric regulation; bacteria; cAMP; CBS domain; cyclic AMP; cystathionine; cystathionine beta-synthase; Cystathionine beta-Synthase - chemistry; Cystathionine beta-Synthase - metabolism; Desulfitobacterium; Enzyme Activation; enzyme activity; fluorescence; Fluorescence Resonance Energy Transfer; Fructose-1-phosphate; inorganic pyrophosphatase; Ligands; moieties; phosphates; Protein Binding; proteins; Pyrophosphatases - chemistry; Pyrophosphatases - metabolism; ribose; Adenosine; Adenine; PPi; CBS-PPase; CBS-protein; CBS domain; cAMP; Fructose-1-phosphate; cpPPase; Fru-1-P; Allosteric regulation; CBS; Mant-AMP; Pi; FRET; dhPPase; PPase
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2019.05.010

Regulatory cystathionine β-synthase (CBS) domains are ubiquitous in proteins, yet their mechanism of regulation remains largely obscure. Inorganic pyrophosphatase which contains regulatory CBS domains as internal inhibitors (CBS-PPase) is activated by ATP and inhibited by AMP and ADP; nucleotide binding to CBS domains and substrate binding to catalytic domains demonstrate positive co-operativity. Methods: Here, we explore the ability of an AMP analogue (cAMP) and four compounds that mimic the constituent parts of the AMP molecule (adenine, adenosine, phosphate, and fructose-1-phosphate) to bind and alter the activity of CBS-PPase from the bacterium Desulfitobacterium hafniense. Adenine, adenosine and cAMP activated CBS-PPase several-fold whereas fructose-1-phosphate inhibited it. Adenine and adenosine binding to dimeric CBS-PPase exhibited high positive co-operativity and markedly increased substrate binding co-operativity. Phosphate bound to CBS-PPase competitively with respect to a fluorescent AMP analogue. Protein interactions with the adenine moiety of AMP induce partial release of the internal inhibition and determine nucleotide-binding co-operativity, whereas interactions with the phosphate group potentiate the internal inhibition and decrease active-site co-operativity. The ribose moiety appears to enhance the activation effect of adenine and suppress its contribution to both types of co-operativity. Our findings demonstrate for the first time that regulation of a CBS-protein (inhibition or activation) is determined by a balance of its interactions with different chemical groups of the nucleotide and can be reversed by their modification. Differential regulation by nucleotides is not uncommon among CBS-proteins, and our findings may thus have a wider significance. •CBS domains of various enzymes and transporters bind regulatory nucleotides•AMP inhibits whereas ATP activates CBS domain-containing pyrophosphatase (PPase)•Adenine, adenosine and cAMP activate PPase whereas fructose-1-phosphate inhibits it•The α-phosphate group and its state determine the sign of the nucleotide effect