A1 Refereed original research article in a scientific journal
Cystathionine β-Synthase (CBS) Domains Confer Multiple Forms of Mg2(+)-dependent Cooperativity to Family II Pyrophosphatases.
Authors: Salminen A, Anashkin V, Lahti M, Tuominen H, Lahti R, Baykov A A
Publisher: The American Society for Biochemistry and Molecular Biology
Publication year: 2014
Journal: Journal of Biological Chemistry
Journal name in source: The Journal of biological chemistry
Journal acronym: J Biol Chem
Volume: 289
Issue: 33
First page : 22865
Last page: 22876
Number of pages: 12
ISSN: 1083-351X
DOI: https://doi.org/10.1074/jbc.M114.589473
Abstract
Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprised of 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 Mg2+, except for the C. novyi PPase, where Mg2+ 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 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.
Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprised of 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 Mg2+, except for the C. novyi PPase, where Mg2+ 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 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.
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