Many prokaryotic soluble PPases (pyrophosphatases) contain a pair of regulatory adenine nucleotide-binding CBS
(cystathionine β-synthase) domains that act as 'internal inhibitors'
whose effect is modulated by nucleotide binding. Although such
regulatory domains are found in important enzymes and transporters, the
underlying regulatory mechanism has only begun to come into focus. We
reported previously that CBS domains bind nucleotides co-operatively and induce positive kinetic co-operativity (non-Michaelian behaviour) in CBS-PPases (CBS
domain-containing PPases). In the present study, we demonstrate that a
homodimeric ehPPase (Ethanoligenens harbinense PPase) containing an
inherent mutation in an otherwise conserved asparagine residue in a loop
near the active site exhibits non-co-operative hydrolysis kinetics. A
similar N312S substitution in 'co-operative' dhPPase (Desulfitobacterium
hafniense PPase) abolished kinetic co-operativity while causing only
minor effects on nucleotide-binding affinity and co-operativity.
However, the substitution reversed the effect of diadenosine
tetraphosphate, abolishing kinetic co-operativity in wild-type dhPPase,
but restoring it in the variant dhPPase. A reverse serine-to-asparagine
replacement restored kinetic co-operativity in ehPPase. Molecular
dynamics simulations revealed that the asparagine substitution resulted
in a change in the hydrogen-bonding pattern around the asparagine
residue and the subunit interface, allowing greater flexibility at the
subunit interface without a marked effect on the overall structure.
These findings identify this asparagine residue as lying at the
'crossroads' of information paths connecting catalytic and regulatory
domains within a subunit and catalytic sites between subunits.