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

Anashkin, Viktor A., Aksenova, Vera A., Vorobyeva, Natalya N., Baykov, Alexander A.
Biochimica et biophysica acta 2019 v.1863 no.8 pp. 1263-1269
Desulfitobacterium, active sites, adenine, adenosine, adenosine diphosphate, adenosine monophosphate, adenosine triphosphate, bacteria, cyclic AMP, cystathionine, cystathionine beta-synthase, enzyme activity, fluorescence, inorganic pyrophosphatase, moieties, phosphates, proteins, ribose
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.