Local pH-dependent conformational changes leading to proteolytic susceptibility of cystatin C

Cystatin C, a cysteine protease inhibitor, was subject to hydrolysis at two sites when complexed with papain and in the presence of excess papain. A pH-dependent cleavage at His-86 increases Asp-87 was observed, as well as a pH-independent one at Gly-4 increases Lys-5. His-86 increases Asp-87 hydrolysis increased with decreasing pH and was characterized kinetically. It could be described by a single ionization with pKa = 3.4 +/- 0.2 and (kcat./Km)max. = 1.4 (+/- 0.4) x 10(4) M-1.s-1 at I = 0.3 M. C.d. spectroscopy, also at I = 0.3 M, demonstrated a conformational change with pKa = 3.2 +/- 0.2, indicating that the pH-dependence of hydrolysis was due to a conformational change in cystatin C. At I = 0.15 M, the pKa of the conformational change observed by c.d. shifted to 4.1 +/- 0.1. This indicates that at physiological ionic strength of 0.15 M, a significant proportion of cystatin C complexed with protease would be in a proteolytically labile conformation over the pH range 4.5 to 5, which is encountered in lysosomes. This may constitute a mechanism for clearing inappropriately localized cystatins. A pH-dependent conformational variability in this region of the inhibitor could explain the differences in the X-ray crystallographic and n.m.r. structures of the homologous chicken cystatin. The ionic-strength dependence of ionization indicates a hydrophobic stabilization of the ionizable group. The lack of pH-dependence of hydrolysis at Gly-4 increases Lys-5, with kcat./Km = 220 +/- 41 M-1.s-1 in the pH range 3.89 to 7.96 was unexpected in light of the normal, bell-shaped pH-dependence of papain-catalysed hydrolyses. This may reflect a different rate-limiting step of cystatin C hydrolysis.

Download Full-text

Metal Ions and Hydrogen Peroxide. XXV

Zeitschrift für Naturforschung B ◽

10.1515/znb-1972-0202 ◽

1972 ◽

Vol 27 (2) ◽

pp. 95-100 ◽

Cited By ~ 6

Author(s):

Peter Waldmeier ◽

Bernhard Prijs ◽

Helmut Sigel

Keyword(s):

Hydrogen Peroxide ◽

Ionic Strength ◽

Metal Ions ◽

Experimental Evidence ◽

Initial Rate ◽

Reaction Order ◽

Ph Dependence ◽

Initial Concentration ◽

Ph Dependent ◽

Ph Range

The decomposition of H2O2, catalyzed by the Co2® complex of 4,4′,4″,4″′-tetrasulfophthalocyanine (CoIIPTS), was investigated in the pH range 3.8 through 10 by measuring the initial rate, v0=d(O2)/dt, of the increasing formation of O2 (25°; I=0.1). In this pH range v0 is proportional to the initial concentration of H2O2 (determined at pH 5.0 and 9.2). Due to the dimerization (log KD=5.47 ±0.09 at natural ionic strength and about 7.63 ±0.16 in 0.1 M NaClO4; 25°) and polymerization of CoIIPTS the catalyst and its reaction order are difficult to establish: Based on the experimental evidence it is suggested that v0 is proportional to the concentration of monomer CoIIPTS. Additionally, there is evidence that the experimentally determined v0 contains the contributions of a pH-independent and a pH-dependent reaction course. These results are analog to those obtained earlier with FeIIIPTS as catalyst. A mechanism for the catalyzed disproportionation of H2O2 by CoIIPTS is proposed. The catalase-like activity of CoIIIPTS (OH) is smaller than that of CoIIPTS and the pH-dependence is different.

Download Full-text

pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

Protein and Peptide Letters ◽

10.2174/0929866526666190617092944 ◽

2019 ◽

Vol 26 (10) ◽

pp. 743-750 ◽

Cited By ~ 1

Author(s):

Remya Radha ◽

Sathyanarayana N. Gummadi

Keyword(s):

Vibrio Cholerae ◽

Conformational Changes ◽

Kinetic Studies ◽

Structural Features ◽

Structure Stability ◽

Kcal Mole ◽

Scanning Calorimetry ◽

Wide Range ◽

Ph Dependent ◽

Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

Download Full-text

Why is the hydrolytic activity of acetylcholinesterase pH dependent? Kinetic study of acetylcholine and acetylthiocholine hydrolysis catalyzed by acetylcholinesterase from electric eel

Zeitschrift für Naturforschung C ◽

10.1515/znc-2017-0134 ◽

2018 ◽

Vol 73 (9-10) ◽

pp. 345-351 ◽

Cited By ~ 2

Author(s):

Alena Komersová ◽

Markéta Kovářová ◽

Karel Komers ◽

Václav Lochař ◽

Alexander Čegan

Keyword(s):

Catalytic Activity ◽

Ionic Strength ◽

Ph Value ◽

Hydrolytic Activity ◽

Initial Substrate ◽

Electric Eel ◽

Value Range ◽

Ph Dependent ◽

Ph Range ◽

Initial Substrate Concentration

AbstractThe dependence of the activity of acetylcholinesterase from electric eel at a pH value range of 4.8–9.8 (phosphate buffer), regarding acetylcholine and acetylthiocholine hydrolysis, was determined at 25 °C, ionic strength of 0.11 M, and initial substrate concentration of 4 mM. At a pH range of 4.8–9.8, the dependencesA(pH) form a sigmoid increasing curve with the maximum catalytic activity at a pH range 8–9.5. For acetylcholine hydrolysis, the kinetic reason for such an increase inAconsists mainly of an increase in the rate constantk2(Michaelis-Menten) model with increasing pH of the reaction mixture. For acetylthiocholine hydrolysis, the kinetic explication of the determined dependenceA(pH) is more complicated.

Download Full-text

A pH-dependent conformational transition of Aβ peptide and physicochemical properties of the conformers in the glial cell

Biochemical Journal ◽

10.1042/bj3610547 ◽

2002 ◽

Vol 361 (3) ◽

pp. 547-556 ◽

Cited By ~ 13

Author(s):

Yoichi MATSUNAGA ◽

Nobuhiro SAITO ◽

Akihiro FUJII ◽

Junichi YOKOTANI ◽

Tadakazu TAKAKURA ◽

...

Keyword(s):

Glial Cell ◽

Conformational Changes ◽

Conformational Transition ◽

Early Stage ◽

Ph Dependence ◽

Amyloid Β ◽

Proteinase K ◽

Aβ Peptide ◽

Ph Dependent ◽

Insight Into

In the present study we identified the epitopes of antibodies against amyloid β-(1–42)-peptide (Aβ1–42): 4G8 reacted with peptides corresponding to residues 17–21, 6F/3D reacted with peptides corresponding to residues 9–14, and anti 5-10 reacted with peptides corresponding to residues 5–10. The study also yielded some insight into the Aβ1–42 structures resulting from differences in pH. An ELISA study using monoclonal antibodies showed that pH-dependent conformational changes occur in the 6F/3D and 4G8 epitopes modified at pH 4.6, but not in the sequences recognized by anti 1-7 and anti 5-10. This was unique to Aβ1–40 and Aβ1–42 and did not occur with Aβ1–16 or Aβ17–42. The reactivity profile of 4G8 was not affected by blockage of histidine residues of pH-modified Aβ1–40 and Aβ1–42 with diethyl pyrocarbonate; however, the mutant [Gln11]Aβ1–40 abrogated the unique pH-dependence towards 4G8 observed with Aβ1–40. These findings suggest that these epitopes are cryptic at pH4.6, and that Glu11 is responsible for the changes. We suggest that the abnormal folding of 6F/3D epitope affected by pH masked the 4G8 epitope. A study of the binding of metal ions to Aβ1–42 suggested that Cu2+ and Zn2+ induced a conformational transition around the 6F/3D region at pH7.4, but did not affect the region when it was modified at pH4.6. However, Fe2+ had no effect, irrespective of pH. Aβ modified at pH 4.6 appeared to be relatively resistant to proteinase K compared with Aβs modified at pH7.4, and the former might be preferentially internalized and accumulated in a human glial cell. Our findings suggest the importance of microenvironmental changes, such as pH, in the early stage of formation of Aβ aggregates in the glial cell.

Download Full-text

Ionic-Strength Dependence of the Conformational Change in the Unphosphorylated Sodium Pump†

Biochemistry ◽

10.1021/bi040018c ◽

2005 ◽

Vol 44 (5) ◽

pp. 1482-1494 ◽

Cited By ~ 2

Author(s):

Shwu-Hwa Lin ◽

Larry D. Faller

Keyword(s):

Ionic Strength ◽

Conformational Change ◽

Sodium Pump ◽

Ionic Strength Dependence ◽

Strength Dependence

Download Full-text

Some Physicochemical Peculiarities of Poplar Plastocyanins a and b

Zeitschrift für Naturforschung C ◽

10.1515/znc-2009-5-617 ◽

2009 ◽

Vol 64 (5-6) ◽

pp. 399-404 ◽

Cited By ~ 2

Author(s):

Petya K. Christova ◽

Anthony A. Donchev ◽

Alexandra C. Shosheva ◽

Vladimir I. Getov ◽

Mitko I. Dimitrov

Keyword(s):

Plant Species ◽

Ph Dependence ◽

Equilibrium Constants ◽

The Other ◽

Redox Potentials ◽

Extinction Coefficients ◽

Structural Differences ◽

Ph Dependent ◽

Ph Range ◽

Reduced Forms

The redox potentials of poplar plastocyanins a and b (PCa, PCb) were determined by spectro photometric titrations of their reduced forms with [Fe(CN)6]3-. It was found that the two isoforms have the following millimolar extinction coefficients ε597, equilibrium constants Keq of one-electron exchange with [Fe(CN)6]4-/[Fe(CN)6]3-, and standard electron potentials E0′: PCa: ε597 = (4.72 ± 0.08) mM-1 cm-1, Keq = 0.133 ± 0.009, E0′ = (354 ± 11) mV; PCb: ε597 = (5.23 ± 0.16) mM-1 cm-1, Keq = 0.175 ± 0.010, E0′ = (363 ± 12) mV. The pH dependence of the redox potential of PCb was studied too. It was found, that the value of E0′ for PCb is constant in the pH range 6.5 - 9.5, but decreases in the range 4.8 - 6.5. On the whole, the dependence resembles that of PC from some well-known plant species, including poplar PCa. The changes of E0′ in the pH-dependent region for poplar PCb, however, are smaller and are 13 mV per pH unit, whereas in the other well-known plant species the changes are about 50 - 60 mV per pH unit. It has been assumed that the weaker pH dependence of E0′ of PCb accounts for some structural differences between PCa and PCb

Download Full-text

Pressure Jump Studies of Proteins. I. Isomerization of Bovine Serum Albumin at Neutral pH

Canadian Journal of Biochemistry ◽

10.1139/o71-183 ◽

1971 ◽

Vol 49 (12) ◽

pp. 1267-1275 ◽

Cited By ~ 15

Author(s):

D. E. Goldsack ◽

P. M. Waern

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Conformational Change ◽

Bovine Serum ◽

Ph Dependence ◽

Kinetic Studies ◽

Relaxation Effect ◽

Pressure Jump ◽

Relaxation Effects ◽

Ph Range

Pressure jump kinetic studies of the conformational change occurring in bovine serum albumin in neutral solutions have been carried out over the pH range 6.5–9.5. Two distinct relaxation effects are observed at each pH. The faster relaxation is attributed to binding of the dye to the protein, and the slower relaxation is related to the conformational change occurring in the protein. This slower relaxation effect is pH dependent with a maximum value near pH 8. Detailed analysis of these data leads to a mechanism for the conformational change which indicates that the one form of the protein has an ionizable group with a pK of 8.7 which changes to a pK of 6.7 when the protein undergoes the conformational change. A simple iterative procedure is given for analyzing the pH dependence of a relaxation time constant for a cyclic mechanism involving only one ionizing group controlling the conformational change.

Download Full-text

pH dependent conformational changes in the T-and R-states of insulin in solution: Circular dichroic studies in the pH range of 6 to 10

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(92)90862-f ◽

1992 ◽

Vol 186 (2) ◽

pp. 1115-1120 ◽

Cited By ~ 6

Author(s):

Yesook Kim ◽

James E. Shields

Keyword(s):

Conformational Changes ◽

Ph Dependent ◽

Ph Range ◽

Circular Dichroic

Download Full-text

Studies on Horseradish Peroxidase. VII. A Kinetic Study of the Oxidation of Iodide by Horseradish Peroxidase Compound II

Canadian Journal of Chemistry ◽

10.1139/v71-511 ◽

1971 ◽

Vol 49 (18) ◽

pp. 3059-3063 ◽

Cited By ~ 24

Author(s):

R. Roman ◽

H. B. Dunford ◽

M. Evett

Keyword(s):

Ionic Strength ◽

Horseradish Peroxidase ◽

Kinetic Study ◽

Rate Constant ◽

Ph Dependence ◽

Order Rate Constant ◽

Acid Dissociation ◽

Ph Range ◽

Compound Ii ◽

Kinetics Of

The kinetics of the oxidation of iodide ion by horseradish peroxidase compound II have been studied as a function of pH at 25° and ionic strength of 0.11. The logarithm of the second-order rate constant decreases linearly from 2.3 × 105 to 0.1 M−1 s−1 with increasing pH over the pH range 2.7 to 9.0. The pH dependence of the reaction is explained in terms of an acid dissociation outside the pH range of the study.

Download Full-text