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

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.

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

Canadian Journal of Biochemistry ◽

10.1139/o81-014 ◽

1981 ◽

Vol 59 (2) ◽

pp. 92-99 ◽

Cited By ~ 3

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.

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

Canadian Journal of Biochemistry ◽

10.1139/o82-109 ◽

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.

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

Canadian Journal of Biochemistry ◽

10.1139/o80-026 ◽

1980 ◽

Vol 58 (3) ◽

pp. 194-200 ◽

Cited By ~ 1

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.

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Slow Conformational Changes of the Inorganic Pyrophosphatase from Bakers' Yeast Induced by Divalent Metal Ions

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1973.tb02686.x ◽

1973 ◽

Vol 33 (2) ◽

pp. 323-331 ◽

Cited By ~ 15

Author(s):

Wolsfgang E. Hohne ◽

Tom A. Rapoport

Keyword(s):

Metal Ions ◽

Conformational Changes ◽

Divalent Metal ◽

Inorganic Pyrophosphatase ◽

Divalent Metal Ions

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Metal derivatives of N1-substituted thiosemicarbazones with divalent metal ions (Ni, Cu): Synthesis and structures

Polyhedron ◽

10.1016/j.poly.2009.12.013 ◽

2010 ◽

Vol 29 (3) ◽

pp. 1130-1136 ◽

Cited By ~ 34

Author(s):

Tarlok S. Lobana ◽

Poonam Kumari ◽

Geeta Hundal ◽

Ray J. Butcher

Keyword(s):

Metal Ions ◽

Divalent Metal ◽

Divalent Metal Ions ◽

Metal Derivatives ◽

Derivatives Of

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Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase

Biological Chemistry ◽

10.1515/hsz-2012-0334 ◽

2013 ◽

Vol 394 (5) ◽

pp. 695-701 ◽

Cited By ~ 2

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.

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

Organic & Biomolecular Chemistry ◽

10.1039/c7ob02001e ◽

2017 ◽

Vol 15 (41) ◽

pp. 8802-8809 ◽

Cited By ~ 2

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.

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