Comparative studies of kinetic and optical properties of rabbit muscle, sturgeon muscle, and yeast pyruvate kinase

1980 ◽  
Vol 58 (3) ◽  
pp. 194-200 ◽  
Author(s):  
Chiu-Yin Kwan ◽  
Jerome L. Gabriel ◽  
Robert C. Davis
Keyword(s):  
Optical Properties ◽  
Metal Ions ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Rabbit Muscle ◽  
Divalent Metal Ions ◽  
Muscle Enzymes ◽  
Muscle Enzyme ◽  
Allosteric Effectors ◽  
Functional Features

The kinetic and optical properties of pyruvate kinase isolated from rabbit muscle, sturgeon muscle, and yeast were compared using various activating divalent metal ions as probes for functional features and using ultraviolet circular dichroism (cd) measurements for conformational features, respectively. All three preparations of pyruvate kinase were similar in many aspects, such as activating efficiencies of the four activating metal ions, Mg(II), Co(II), Mn(II), and Ni(II) and pH-rate profiles, suggesting the presence of a similar metal binding locus of these enzymes as well as a common underlying mechanism of action. L-Phe inhibited the rabbit muscle enzyme and turned the hyperbolic kinetics into a sigmoidal kinetic with respect to phosphoenolpyruvate at alkaline pH, while fructose-1,6-biphosphate activated the sturgeon muscle and yeast enzymes and turned the sigmoidal kinetics into hyperbolic kinetics with respect to phosphoenolpyruvate. The ultraviolet cd spectral changes qualitatively correlated well with kinetic observations of all three native enzymes in the presence and absence of allosteric effectors. Our results suggested that there are at least two conformational states of pyruvate kinase which are inducible by the binding of substrate and (or) allosteric effectors. The conformational changes from one form to another in these enzymes are very similar, especially between the rabbit and sturgeon muscle enzymes.

pH-Dependent amino acid induced conformational changes of rabbit muscle pyruvate kinase

1980 ◽  
Vol 58 (3) ◽  
pp. 188-193 ◽  
Author(s):  
Chiu-Yin Kwan ◽  
Robert C. Davis
Keyword(s):  
Metal Ions ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Divalent Metal ◽  
Rabbit Muscle ◽  
Divalent Metal Ions ◽  
Spectral Changes ◽  
Metal Derivatives ◽  
Muscle Pyruvate Kinase ◽  
Difference Absorption

The interactions of L-Phe and L-Ala with rabbit muscle pyruvate kinase depended upon the nature of divalent metal ions studied: Mg(II), Co(II), Mn(II), and Ni(II). L-Phe inhibited all metal derivatives of the enzyme except Mn(II)–enzyme. L-Ala inhibited only Ni(II)–enzyme and had no effect on other metal derivatives. The inhibition by L-Phe could be partially or completely reversed by L-Ala for all metal derivatives. The mode of inhibition of pyruvate kinase by L-Phe depended upon pH as well as the nature of activating divalent metal ions. The sigmoidal response increased with increasing pH for all metal derivatives inhibited by L-Phe. L-Phe and L-Ala strongly perturbed the coordination sphere of enzyme bound Co(II), but not Ni(II). There were poor correlations between visible circular dichroic (cd) spectral changes and the corresponding kinetic changes. However, L-Phe and (or) L-Ala induced ultraviolet cd and difference absorption spectral changes, on the other hand, corresponded remarkably well with the kinetic observations.

Interaction of D-phenylalanine with Co(II)-substituted rabbit muscle pyruvate kinase: kinetic and optical properties

1982 ◽  
Vol 60 (9) ◽  
pp. 861-866
Author(s):  
Chiu-Yin Kwan ◽  
Robert C. Davis
Keyword(s):  
Amino Acids ◽  
Optical Properties ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Metal Ion ◽  
Inhibitory Effect ◽  
Rabbit Muscle ◽  
Divalent Metal Ion ◽  
Muscle Pyruvate Kinase ◽  
Difference Absorption

The kinetic and optical properties of Co(II)-substituted pyruvate kinase in the presence of D-phenylalanine (D-Phe) were investigated. The results are discussed in comparison with the effects of its optical isomer L-phenylalanine (L-Phe) on the same enzyme. The catalytic effect of D-Phe on rabbit muscle pyruvate kinase depended upon the nature of the activating divalent metal ion used. It has stimulatory effect on Mg(II)-activated enzyme, but inhibitory effect on Co(II)-activated enzyme. Unlike the inhibitory effect of L-Phe, the inhibition of Co(II)–enzyme by D-Phe was not sensitive to the changes of pH and temperature, could not be reversed by L-alanine (L-Ala), displayed hyperbolic kinetics, and was noncompetitive with respect to phosphoenolpyruvate saturation. D-Phe induced substantial visible circular dichroism (CD) spectral changes of Co(II)–enzyme similar to those induced by L-Phe. Although ultraviolet CD spectrum was not affected, D-Phe induced an ultraviolet difference absorption spectral change very similar to, but much smaller than, that induced by L-Phe. Our results support that D-Phe and other amino acids interact with the enzyme at two different sites: a common site, causing similar conformational changes which bear little direct kinetic relevance, and a kinetically relevant site, which is sterically dependent upon the side chain of the amino acids.

L-Phenylalanine induced changes of sulfhydryl reactivity in rabbit muscle pyruvate kinase

1981 ◽  
Vol 59 (2) ◽  
pp. 92-99 ◽  
Author(s):  
Chiu-Yin Kwan ◽  
Robert C. Davis
Keyword(s):  
Metal Ions ◽  
Pyruvate Kinase ◽  
Divalent Metal ◽  
Enzyme Inactivation ◽  
Rabbit Muscle ◽  
Divalent Metal Ions ◽  
Substrate Analogue ◽  
Metal Derivatives ◽  
Sulfhydryl Modification ◽  
Muscle Pyruvate Kinase

Reactivity of sulfhydryl groups in rabbit muscle pyruvate kinase toward 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was studied in the presence of activating divalent metal ions, substrate, substrate analogue, and the allosteric inhibitor, L-Phe. The pattern of sulfhydryl modification in various complexes of pyruvate kinase was consistent with the extent of enzyme inactivation by DTNB under very similar conditions. The sulfhydryl reactivity of Mg(II)-, Co(II)-, and Mn(II)-substituted pyruvate kinase toward DTNB depended upon the nature of the activating divalent metal ions used in the following order of increasing potency, Mg(II) < Mn(II) < Co(II), which is inversely related to the order of catalytic efficiency of these metal ions at alkaline pH. Similar optical spectra and the patterns of sulfhydryl modification by DTNB of the metal derivatives of pyruvate kinase were observed upon the binding of the substrate, phosphoenolpyruvate (PEP), or the substrate analogue, phosphoglycolate, which also provided a complete protection against enzyme inactivation by DTNB. L-Phe, on the other hand, deprotected the enzyme from inactivation and further sulfhydryl modification by DTNB in the presence of PEP with the following order of potency depending upon the activating metal ions, Mn(II) < Co(II) < Mg(II), which parallels the order of metal dependency of L-Phe inhibition of this enzyme. L-Ala, which reverses the L-Phe inhibition of Mg(II)- or Co(II)-activated enzyme, restored the protective effect of PEP in the presence of L-Phe. The different patterns of sulfhydryl reactivity toward Mn(II)–enzyme (hyperbolic) and Mg(II)–enzyme (sigmoidal) correspond well with their kinetic patterns in the presence of L-Phe, indicating the presence of different conformational states between these two metal–enzyme complexes. These results led us to conclude that enzyme sulfhydryl reactivity toward DTNB can be used as a valid index for allosteric conformational changes of rabbit muscle pyruvate kinase.

scholarly journals Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase

1973 ◽  
Vol 135 (2) ◽  
pp. 265-276 ◽  
Author(s):  
B. Focant ◽  
D. C. Watts
Keyword(s):  
Creatine Kinase ◽  
Smooth Muscle ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Intermediate Stage ◽  
Hill Coefficient ◽  
Rabbit Muscle ◽  
Difference Spectra ◽  
Muscle Creatine Kinase ◽  
Quaternary Complex

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO3- or Cl- ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by αβ-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO3- ions to the enzyme–creatine–MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme–creatine–NO3-–MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO3- is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme–pyruvate–NO3-–MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or α-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K+-free enzyme. Only the former inhibits the K+-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.

Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase

2013 ◽  
Vol 394 (5) ◽  
pp. 695-701 ◽  
Author(s):  
Christian Boehme ◽  
Frank Bieber ◽  
Julia Linnemann ◽  
Reinhard Breitling ◽  
Stefan Lorkowski ◽  
...  
Keyword(s):  
Metal Ions ◽  
Pyruvate Kinase ◽  
Adenosine Diphosphate ◽  
Transition Metal Ions ◽  
Recombinant Enzyme ◽  
High Yield ◽  
Rabbit Muscle ◽  
Reading Frame ◽  
Ph Range ◽  
Muscle Pyruvate Kinase

Abstract The stepwise synthesis of thymidine triphosphate (TTP) requires a kinase for phosphorylation in the last step. Because pyruvate kinase (PK) using phosphoenolpyruvate (PEP) as substrate can regenerate adenosine triphosphate and phosphorylate thymidine diphosphate as well, we chose this enzyme for the synthesis of TTP via an enzymatic cascade reaction. The metalloenzyme PK shows pronounced promiscuity and therefore fits well to the conditions of this reaction. PK commonly used today is isolated from rabbit muscle. We cloned and expressed the respective open reading frame in Escherichia coli, purified, and characterized the His-tagged recombinant enzyme. The enzyme has an activity optimum at 37°C and in the pH range from 7.4 to 7.8. Km constants conformed well with the isolated native enzyme for adenosine diphosphate (ADP) to 0.37±0.02 mm and for PEP to 0.07±0.01 mm. The recombinant enzyme shows the following range in its substrate specificity: ADP>dADP>dGDP>dCDP>thymidine diphosphate (TDP). It allows the phosphorylation of TDP to TTP in high yield (up to 95%). The metal ions Mg2+ and K+ are necessary for full enzymatic activity. The addition of transition metal ions such as Mn2+, Cu2+, Co2+, and Ni2+ reduces activity. Storage of the enzyme at -20°C retains full activity.

Local conformational changes in the 8–17 deoxyribozyme core induced by activating and inactivating divalent metal ions

2017 ◽  
Vol 15 (41) ◽  
pp. 8802-8809 ◽  
Author(s):  
Alessio Peracchi ◽  
Maria Bonaccio ◽  
Alfredo Credali
Keyword(s):  
Metal Ions ◽  
Conformational Change ◽  
Conformational Changes ◽  
Divalent Metal ◽  
Divalent Metal Ions ◽  
Specific Metal

Placing 2-aminopurine at position 15 of the 8–17 DNAzyme allows the detection of a specific metal-induced conformational change, apparently coupled to the activation of catalysis.

Nanopore analysis of the interaction of metal ions with prion proteins and peptidesThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (2) ◽  
pp. 347-358 ◽  
Author(s):  
Radu I. Stefureac ◽  
Claudia Avis Madampage ◽  
Olga Andrievskaia ◽  
Jeremy S. Lee
Keyword(s):  
Metal Ions ◽  
Conformational Changes ◽  
Metal Ion ◽  
Prion Proteins ◽  
Peer Review Process ◽  
Cellular Biology ◽  
Divalent Metal Ions ◽  
High Concentrations ◽  
Individual Peptide ◽  
Selection Of

Nanopore analysis can be used to study conformational changes in individual peptide or protein molecules. Under an applied voltage there is a change in the event parameters of blockade current or time when a molecule bumps into or translocates through the pore. If a molecule undergoes a conformational change upon binding a ligand or metal ion the event parameters will be altered. The objective of this research was to demonstrate that the conformation of the prion protein (PrP) and prion peptides can be modulated by binding divalent metal ions. Peptides from the octarepeat region (Octa2, (PHGGGWGQ)2 and Octa 4, (PHGGGWGQ)4), residues 106–126 (PrP106–126), and the full-length Bovine recombinant prion (BrecPrP) were studied with an α-hemolysin pore. Octa2 readily translocated the pore but significant bumping events occurred on addition of Cu(II) and to a lesser extent Zn(II), demonstrating that complex formation was occurring with concomitant conformational changes. The binding of Cu(II) to Octa4 was more pronounced and at high concentrations only a small proportion of the complex could translocate. Addition of Zn(II) also caused significant changes to the event parameters but Mg(II) and Mn(II) were inert. Addition of Cu(II) to PrP106–126 caused the formation of a very tight complex, which could not translocate the pore. Small changes were observed with Zn(II), but not with Mg(II) or Mn(II). Analysis of BrecPrP showed that about 37% were translocation events, but on addition of Cu(II) or Zn(II) these disappeared and only bumping events were recorded. Suprisingly, addition of Mn(II) caused an increase in translocation events to about 64%. Thus, conformational changes to prions upon binding metal ions are readily observed by nanopore analysis.

scholarly journals Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

Biochemistry ◽  
2015 ◽  
Vol 54 (41) ◽  
pp. 6369-6381 ◽  
Author(s):  
Aamir Mir ◽  
Ji Chen ◽  
Kyle Robinson ◽  
Emma Lendy ◽  
Jaclyn Goodman ◽  
...  
Keyword(s):  
Metal Ions ◽  
Conformational Changes ◽  
Hammerhead Ribozyme ◽  
Divalent Metal ◽  
Divalent Metal Ions ◽  
Cleavage Reaction
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