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.