Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations. Study holds ProTherm entries: 18643, 18644, 18645, 18646, 18647, 18648, 18649, 18650, 18651, 18652, 18653, 18654, 18655, 18656, 18657, 18658, 18659, 18660, 18661, 18662, 18663, 18664, 18665, 18666, 18667, 18668, 18669, 18670, 18671 Extra Details: Enzymes were preincubated with 1.5 mM Mn2+; 13 microM EGTA was added in the experiment Mn(2+) binding affinity; thermostability; active site structure; conservative mutations
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:50 p.m.
|Number of data points||29|
|Proteins||Manganese-dependent inorganic pyrophosphatase ; Manganese-dependent inorganic pyrophosphatase|
|Assays/Quantities/Protocols||Experimental Assay: Tm details:Additives EDTA (2 mM), ; Experimental Assay: Tm details:Additives EGTA (130 microM), ; Experimental Assay: Tm details:Additives EGTA (13 microM),|
|Libraries||Mutations for sequence MEKILIFGHQNPDTDTICSAIAYADLKNKLGFNAEPVRLGQVNGETQYALDYFKQESPRLVETAANEVNGVILVDHNERQQSIKDIEEVQVLEVIDHHRIANFETAEPLYYRAEPVGCTATILNKMYKENNVKIEKEIAGLMLSAIISDSLLFKSPTCTDQDVAAAKELAEIAGVDAEEYGLNMLKAGADLSKKTVEELISLDAKEFTLGSKKVEIAQVNTVDIEDVKKRQAELEAVISKVVAEKNLDLFLLVITDILENDSLALAIGNEAAKVEKAFNVTLENNTALLKGVVSRKKQVVPVLTDAMAE|