Role of bound calcium ions in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability

Voordouw, Gerrit; Milo, Carol; Roche, Rodney S.

doi:10.1021/bi00662a012

Cited by 149 publications

(90 citation statements)

References 21 publications

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“…7), probably due to immobilization of the phosphate anions and the charged side chains they bind. Similar large negative entropy and enthalpy changes have been observed upon addition of Ca2+ to calcium-binding proteins (Voordouw et al, 1976). …”

Section: Effect Of Phosphatesupporting

confidence: 69%

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Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Wilcox

Eisenberg

1992

Protein Science

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The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an a-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 "C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pK, values of 7.5 and 8. [7][8][9][10][11][12][13][14][15][16][17][18] agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 "C depending on the pH, unfolding above and below that temperature. AC; for folding ranges from -0.085 to -0.102 cal g" K-', comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). A H o and ASo are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, w., 1959, Adv. Protein Chem.14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 "C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 "C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system. Abbreviations: CD, circular dichroism; far UV, 260-185 nm; near UV, 330-245 nm; AzgO, light absorption at 280 nm; AC:, the standard change in heat capacity from the unfolded to the folded form; At = AL -AR, where AL and AR are the absorbance of left and right circularly polarized light, respectively; AcZU, Ac at 222 nm; AtZB2, At at 282 nm; Th, a temperature at which AHo(T) = 0; T,, a temperature at gates to form an a-helical tetramer. The amount of tetwhich ASo( T ) = 0; TES, N-tris(hydroxyrnethyl)methyl-2-aminoethane sulfonic acid. Keywords

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Section: Effect Of Phosphatesupporting

confidence: 69%

“…A similar effect with divalent cations (and negative charges) has been noted in other protein systems (Voordouw et al, 1976;Filimonov et al, 1978;Bashford et al, 1986;Linse et al, 1988;Pantoliano et al, 1988). The effect of phosphate above its second pK, is expected to be essentially the same as sulfate.…”

Section: Effect Of Phosphatesupporting

confidence: 58%

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Wilcox

Eisenberg

1992

Protein Science

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“…In contrast to calcium-saturated Fpp1, which lost one-half of its activity after approximately 25 min of incubation at 40°C, the calciumfree enzyme underwent complete autolysis after 2 min of incubation at 40°C. Thus, this biochemical behavior of Fpp1 is similar to the behavior of the metalloproteases produced by P. fluorescens (34), which required Ca 2ϩ ions for activity and/or thermal stability, and to the behavior of the subtilisin family of proteases, which are calcium-dependent serine proteases (52,54). Of these enzymes, subtilisin (52), proteinase K (1), the calcium-stimulated protease from the cyanobacterium Anabaena variabilis (36), aqualisin from Thermus aquaticus (35), and the cell envelope proteinase from Lactococcus lactis (14,15) are probably the best studied.…”

Section: Discussionsupporting

confidence: 52%

Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen Flavobacterium psychrophilum

Secades

Álvarez

Guijarro

2001

Appl Environ Microbiol

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Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (F. psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

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“…The known difference between TRM and SCB [3,5] in intrinsic stability is confirmed. This difference is smaller if enzyme species are compared with identical calcium ion content.…”

Section: Methodsmentioning

confidence: 55%

“…Examples are trypsin, thermolysin, subtilisin and others [1][2][3][4][5][6][7][8][9][10][11]. In the subtilisin family, subtilisin Carlsberg (SCB), subtilisin BPN', proteinase K and thermitase (TRM), respectively, can bind Ca 2÷.…”

Section: Introductionmentioning

confidence: 99%

Calcium ion binding by thermitase

Briedigkeit

Frömmel

1989

FEBS Letters

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Thermitase is a thermostable member of the subtilisin protease family. It was found that this enzyme binds very strongly one calcium ion. This metal ion cannot be removed without denaturation by any known method. A second binding site for calcium ions can be depleted partially by gel filtration, similar to the strong binding site of subtilisin Carlsberg. In both enzymes the calcium ion bound to this site can be removed completely using Ca 2÷ chelating reagent. In thermitase and subtilisin Carlsberg, depleting calcium ions from any binding site results in a significant decrease of stability against autolysis.

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Role of bound calcium ions in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability

Cited by 149 publications

References 21 publications

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen Flavobacterium psychrophilum

Calcium ion binding by thermitase

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