title: Cystathionine β-synthase (CBS) domains confer multiple forms of Mg2+-dependent cooperativity to family II pyrophosphatases.
abstract: Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine β-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (Km) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg(2+) except for the C. novyi PPase where Mg(2+) produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller Km value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PPi level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.
authors.affiliation: Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.
authors.forename: Anu
authors.initials: A
authors.lastname: Salminen
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia.
authors.forename: Viktor A
authors.initials: VA
authors.lastname: Anashkin
authors.affiliation: Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.
authors.forename: Matti
authors.initials: M
authors.lastname: Lahti
authors.affiliation: Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.
authors.forename: Heidi K
authors.initials: HK
authors.lastname: Tuominen
authors.affiliation: Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and. Electronic address: reijo.lahti@utu.fi.
authors.forename: Reijo
authors.initials: R
authors.lastname: Lahti
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia. Electronic address: baykov@genebee.msu.su.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011755:Pyrophosphatases; D003541:Cystathionine beta-Synthase
keywords: Allosteric Regulation; Bacterial Signal Transduction; CBS Domain; Cooperativity; Enzyme Inhibitor; Ligand-binding Protein; Pyrophosphatase