title: An asparagine residue mediates intramolecular communication in nucleotide-regulated pyrophosphatase.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, 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.
authors.forename: Anu
authors.initials: A
authors.lastname: Salminen
authors.affiliation: Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia.
authors.forename: Natalia N
authors.initials: NN
authors.lastname: Vorobjeva
authors.affiliation: Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland reijo.lahti@utu.fi baykov@genebee.msu.su.
authors.forename: Reijo
authors.initials: R
authors.lastname: Lahti
authors.affiliation: Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia reijo.lahti@utu.fi baykov@genebee.msu.su.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
mesh_terms: D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003541:Cystathionine beta-Synthase / Q000737:chemistry*; D006094:Gram-Positive Bacteria / Q000201:enzymology; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D001216:Asparagine; D011755:Pyrophosphatases; D003541:Cystathionine beta-Synthase
keywords: CBS domain; co-operativity; molecular dynamics; nucleotide regulation; pyrophosphatase