3.4.21.62 Ca2+ - 688387, 689851, 690033 3.4.21.62 Ca2+ as found in other Bacillus subtilisins, the structure of wild-type Savinase contains 2 calcium ion-binding sites 29435 3.4.21.62 Ca2+ bound calcium ions play a key role in protecting against autolysis and thermal denaturation 649692, 652805 3.4.21.62 Ca2+ Ca2+-binding loop is required for folding of subtilisin but does not seriously contribute to the stabilization of subtilisin in a native structure 707480 3.4.21.62 Ca2+ calcium ion binds weakly to the Ca-7 site in the unautoprocessed form, but is trapped upon autoprocessing. The Ca-7 site is required to promote the autoprocessing reaction by stabilizing the autoprocessed form, in which the new N-terminus of the mature domain is structurally disordered 688371 3.4.21.62 Ca2+ calcium-loaded state of five ions bound to each of the two subtilisin molecules. Three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion 710507 3.4.21.62 Ca2+ enzyme activity and/or stability depends on the presence of divalent cations, probably Ca2+ ions, near the surface of the enzyme 707260 3.4.21.62 Ca2+ five binding sites, important for correct folding 678747 3.4.21.62 Ca2+ importance of calcium in the medium after enzyme induction, both for stability of the proteinase and cell health. The two calcium binding sites have apparent binding constants in the mM range. Binding of calcium to the weaker of those two sites only affects resistance of the enzyme against irreversible thermal inactivation. Kinetics 752829 3.4.21.62 Ca2+ in the presence of calcium the half-life at 55°C and 60°C are 3.6fold and 3.48fold higher for the native enzyme compared to that in the absence of added calcium. In the presence of 10 mM calcium the half-life of the enzyme at 60°C increases by 6.06fold, 5.20fold and 2.92fold when coupled with oxidized sucrose polymers OSP400, OSP70 and polyglutaraldehyde, respectively 686432 3.4.21.62 Ca2+ increase on activity by 20% 667756 3.4.21.62 Ca2+ is important for ISP activity through structural changes. The looped turn constituted by residues Ala180-Pro197 binds Ca2+. Removal of Ca2+ at sites close to the dimer interface and the S1 pocket are involved enzyme inhhibition by EDTA 731819 3.4.21.62 Ca2+ is required for catalysis 706989 3.4.21.62 Ca2+ required for catalysis 706989 3.4.21.62 Ca2+ required for thermostability 731332 3.4.21.62 Ca2+ required, the enzyme sequence contains a low-affinity Ca2+ binding site 752524 3.4.21.62 Ca2+ required, the presence of a low-affinity Ca2+ binding site formed by the backbone carbonyls cannot be excluded based on the amino acid sequence comparisons 731172 3.4.21.62 Ca2+ six calcium ions bind to pro-S255A at the loop regions. The at least six Ca2+ ions bind to pro-S255A too tightly to be removed by dialysis 684164 3.4.21.62 Ca2+ stabilizing mutations are usually calcium-dependent in their stabilizing effect 650018 3.4.21.62 Ca2+ the enzyme contains 7 Ca2+ ions, 4 of which are responsible for folding requirement of Ca2+ ions for the hyperthermostability of Tk-subtilisin, the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin 726988 3.4.21.62 Ca2+ three binding sites, important for correct folding 678747 3.4.21.62 Ca2+ two binding sites, important for correct folding 678747 3.4.21.62 Cd2+ stabilizes 649692 3.4.21.62 Cl- treatment with CsCl increases the activity several fold. Two Cl- ions are close to the mouth of the active site cleft, where they may affect catalysis 708884 3.4.21.62 Cs+ treatment with CsCl increases the activity several fold. Two Cs+ ions are close to the mouth of the active site cleft, where they may affect catalysis 708884 3.4.21.62 Cu2+ 100% inhibition 717205 3.4.21.62 Cu2+ 5 mM, 40% increase in activity 669738 3.4.21.62 KCl increases enzymatic activity somewhat, while choline-Cl has a larger effect 708884 3.4.21.62 Mg2+ 100% inhibition 717205 3.4.21.62 Mg2+ required 731172, 752524 3.4.21.62 additional information enzymatic activity of subtilisin crystals in acetonitrile is sensitive to the type of counterions present before transfer to the organic solvent. Larger cations increase the enzyme activity 708884 3.4.21.62 additional information enzyme contains no metal ion 29425 3.4.21.62 additional information metal ion sites are not formed until late in folding and do not influence the kinetics of folding to the intermediate complex 685161 3.4.21.62 additional information metal ions are not involved in the subtilisin catalytic mechanism but are an important structural element 731819 3.4.21.62 additional information shows no evidence of Ca2+ activation 649830 3.4.21.62 additional information strain DJ-4, not significantly affected by Ca2+, Co2+, Mg2+, or Mn2+ 650609 3.4.21.62 additional information unlike subtilisin, subtilisin-like serine protease does not require Ca2+ for folding 710470 3.4.21.62 Sr2+ increases thermostability, but to a significantly lower degree than Ca2+ 649692