Effects of Active Site Mutations on the Metal Binding Affinity, Catalytic Competence, and Stability of the Family II Pyrophosphatase from Bacillus subtilis. Export

@article{citeulike:120007, abstract = {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.}, address = {Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland, A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia, and Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland.}, author = {Halonen, Pasi and Tammenkoski, Marko and Niiranen, Laila and Huopalahti, Sauli and Parfenyev, Alexey N. and Goldman, Adrian and Baykov, Alexander and Lahti, Reijo}, citeulike-article-id = {120007}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/bi047926u}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/bi047926u}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/15751976}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=15751976}, day = {15}, doi = {10.1021/bi047926u}, issn = {0006-2960}, journal = {Biochemistry}, keywords = {bacillus-subtilis, family-ii-pyrophosphatase}, month = {March}, number = {10}, pages = {4004--4010}, posted-at = {2005-03-10 10:26:12}, priority = {2}, title = {Effects of Active Site Mutations on the Metal Binding Affinity, Catalytic Competence, and Stability of the Family II Pyrophosphatase from Bacillus subtilis.}, url = {http://dx.doi.org/10.1021/bi047926u}, volume = {44}, year = {2005} }

TY - JOUR ID - citeulike:120007 L3 - citeulike-article-id:120007 N2 - 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. IS - 10 JF - Biochemistry SN - 0006-2960 AD - Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland, A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia, and Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland. EP - 4010 TI - Effects of Active Site Mutations on the Metal Binding Affinity, Catalytic Competence, and Stability of the Family II Pyrophosphatase from Bacillus subtilis. VL - 44 SP - 4004 KW - bacillus-subtilis KW - family-ii-pyrophosphatase AU - Halonen, Pasi AU - Tammenkoski, Marko AU - Niiranen, Laila AU - Huopalahti, Sauli AU - Parfenyev, Alexey AU - Goldman, Adrian AU - Baykov, Alexander AU - Lahti, Reijo PY - 2005/03/15/ UR - http://dx.doi.org/10.1021/bi047926u ER -