Spectrophotometric investigations of the interaction of native and chemically modified ATP:guanidinophosphotransferases with their substrates

Roustan, Claude; Pradel, Louise‐Anne; Kassab, Rhida; Fattoum, Abdellatif; Thoại, Nguyễn Văn

doi:10.1016/0005-2744(70)90153-1

Cited by 45 publications

(26 citation statements)

References 25 publications

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“…It would not, of course, necessarily involve a direct interaction between NO3-and the tyrosine residue. Finally, this observation explains why a similar effect of NO3-is not found with rabbit muscle creatine kinase, since, as shown by Roustan et al (1970), creatine kinase does not have a similarly accessible tyrosine residue.…”

Section: Discussionsupporting

confidence: 59%

“…7) is ofconsiderable interest in the light of the finding by Fattoum et al (1971) that this enzyme is inactivated by iodination or nitration of a single tyrosine residue, and that enzyme so modified no longer gives a difference spectrum with either substrate (Roustan et at., 1970). It has been suggested (Landon et al, 1970) that this tyrosine residue is located in a helical region of the enzyme, a great part of which is exposed to the solvent.…”

Section: Discussionmentioning

confidence: 99%

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Effects of anions on a monomeric and a dimeric arginine kinase

Anosike

Watts

1975

Biochemical Journal

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1. Some effects of anions on the rates of phosphoarginine synthesis by monomeric (lobster) and by dimeric (Holothuria forskali) arginine kinases are reported. 2. As with creatine kinase, acetate ions activate both enzymes: C1-was also found to activate both although this was an inhibitor of creatine kinase. 3. NO3-inhibits the lobster enzyme.Inhibition is of the mixed type with respect to MgATP. K1 >K1' and K, >IK.' indicating that the presence of N03-promotes the binding of substrate and vice versa. 4. NO3-alone has no effect on the difference spectrum of the lobster enzyme but in the presence of arginine, MgATP, MgADP, MgAMP or MgIDP the difference spectrum is greatly enhanced. A profound effect on the ionization state oftyrosine residues is inferred. 5. With the Holothuria enzymelow concentrations ofNO3-activate in a manner that iscompetitive with arginine. Higher concentrations cause inhibition of the mixed type with respect to arginine in a similar manner to that found with MgATP for the lobster kinase. 6. Ofa range of anions tested only NO3-and NO2-enhanced the inhibition of enzyme activity by MgADP, indicating the formation of a pseudo-transition-state dead-end complex, enzyme-arginine-NO3-MgADP. The effect was essentially independent of temperature with the Holothuria enzyme, but with the lobster enzyme was much less marked and temperature dependent. The difference may reflect the different stabilities of the monomer and dimer enzymes, although with neither arginine kinase is the stabilization of the dead-end complex as marked as is found with creatinine kinase.Mitner-White & Watts (1971) found tha-t a strong inhibition of rabbit muscle creatine kinase was caused by NO3-ions in the presence of the dead-end complex, creatine-enzyme-MgADP. It was suggested that the planar anion acted by mimicking the transferable phosphoryl group in a transition stage in the reaction and thereby locked the two substrates together. The effectiveness of other anions as inhibitors was related to the extent to which they could behave like NO3-ions. Detailed protonrelaxation-rate studies by Cohn and co-workers confirmed and extended this hypothesis (McLaughlin & Cohn, 1972;, and recently further support has come from investigations with the fluorescent probe 8-anilinonaphthalene-1-sulphonate (McLaughlin, 1974 The work reported below was designed to discover whether arginine kinases were inhibited by low concentrations of anions, lOOmm and less, in a similar manner to the creatine kinases. With two arginine kinases, the monomeric form from the lobster, Homarus vulgaris, and the dimeric form from the sea cucumber, Holothuria forskali, evidence has been presented to indicate that stabilization of a substrate dead-end complex could occur. However, the picture is complicated by additional protein-ion interactions. Materials and MethodsADP (disodium salt) and ATP (disodium salt) were

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Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Effects of anions on a monomeric and a dimeric arginine kinase

Anosike

Watts

1975

Biochemical Journal

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“…The difference spectra displayed by the interaction of substrates with native and modified phosphagen kinases afford some information about the structures of their active sites and allow us to distinguish between one catalytic and two binding sites [1,2].…”

Section: Discussionmentioning

confidence: 99%

Structural Properties of the Creatine‐Kinase Active Site Studied by Chromophoric‐Reagent Labelling

Roustan

Brevet

Pradel

1973

European Journal of Biochemistry

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The environment of the active site of creatine kinase has been investigated by means of three chromophoric reagents : 2-hydroxy-5-nitrobenzyl bromide, 2-methoxy-5-nitrobenzyl bromide and 2-(dansy1amino)ethyl monophosphate.1. The first two reagents are incorporated at the essential thiol groups. The effect of pH on the spectra of the 2-hydroxy-5-nitrobenzyl-labelled creatine kinase suggests that this reagent lies inside the active site near an ionized residue, while the 2-methoxy-5-nitrobenzyl group is apparently exposed to the medium. Conformational changes induced by the binding of different ligands, including nucleotide substrates or anions, produce only perturbations of the 2-hydroxy-5-nitrobenzyl chromophore.2. The 2-(dansy1amino)ethyl monophosphate (which is really an analogue of the nucleotide substrate) interacts with the nucleotide site of creatine and arginine kinases in a relatively hydrophobic environment.The study of the spectral changes induced by the binding of substrates in the environment of intrinsic chromophores such as tyrosyl, cysteinyl or tryptophanyl residues has provided an excellent means for the study of the active site of phosphagen kinases.In previous papers [i, 21, we have reported that the binding of nucleotide substrates to a specific site of creatine kinase induces small conformational changes in the environment of the binding site. Such conformational changes have been also reported elsewhere [3][4][5][6][7][8][9][10] and studied by means of kinetic methods, hydrogen-deuterium exchange, magnetic resonance and optical rotatory dispersion.I n many investigations, "reporter groups" have been used with success to study the microenvironment of the active sites of enzymes such as chymo- bromide (reagent I), 2-methoxy-5-nitrobenxyl bromide (reagent 11) and 2-(dansy1amino)ethyl monophosphate. MATERIALS AND METHODS MATERIALS1 -Dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride) was a product of the Pierce Chemical Co. Nucleotides were obtained from Calbiochem (A grade). All the nucleotide solutions were adjusted to pH 7 by addition of NaOH. Koshland reagent I (2-hydroxy-5-nitrobenzyl bromide) was purchased from Pierce Chemical Co. and dissolved in acetone. Sodium salt of Koshland reagent I was prepared according to [16] by dissolving the product in boiling water and adding sodium carbonate until effervescence stopped. The sodium salt was then evaporated, recrystallized in water and dried a t 100 "C-Koshland reagent I1 (2-methoxy-5-nitrobenzyl bromide) was purchased from Sigma and dissolved in acetone. 2-(Dansy1amino)ethyl monophosphate was synthesized by a modification of the method of Onodera and Yagi [17]. Instead of dansylation of aminoethanol followed by phosphorylation, the dansylation was performed according to the Gray procedure [18] on 2-aminoethylphosphate, first synthesized as described by Fischer et al. [19]. Purification of (dansy1amino)- Eur. J. Biochem. 39 (1973)

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“…Since the preparation of lombricine kinase used in the present work can be estimated to be at least 95 pure, according to the different analytical tests of purity, a contaminant protein fraction cannot account for the values obtained from spectrophotometric titration of the number of nucleotide substrate binding sites and essential sulfhydryl groups [2,3]. Thus it seems reasonable to assume that the two polypeptide chains of lombricine kinase are not identical although attempts to characterize them either by analytical polyacrylamide gel electrophoresis in 8 M urea in alkaline and acidic buffers, or by fingerprints, were unsuccessful.…”

Section: Separation Of the Essential -Sh-containing Tryptic Peptide Fmentioning

confidence: 99%

“…OnIy one binding site for the nucleotide substrate ADP-Mg and only one essential thiol group could be determined per mole of enzyme [2,3]. This raises the question of whether the chemical structures within the two subunits of lombricine kinase are identical or not, or whether a conformational change which occurs either on binding the first mole of ADPMg or one mole of -SH inhibitor on one subunit allows the other to become mireactive.…”

mentioning

confidence: 99%

Separation of the Two Non‐Identical Subunits of Lombricine Kinase from Lumbricus terrestris Muscle by Chromatography on Sepharose‐Mercurial

Terrossian¹,

Pradel²,

Kassab³

et al. 1974

European Journal of Biochemistry

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Of the dimeric phosphagen kinases (M, = 80000) studied, lombricine kinase is the only one which contains only one essential thiol group and one binding site for ADP-Mg. These facts suggest a non-identity between the two subunits of this enzyme.The reversible labeling of the essential thiol group of this protein with 2,4-dinitrofluorobenzene, followed by alkylation of the remaining thiol groups, allowed the separation of the two subunits by means of a Sepharose-mercurial column. The Sepharose-mercurial was prepared by treatment of Sepharose 4B with p-aminophenyl mercuric acetate.Amino-acid analyses, N-and C-terminal determinations and fingerprintings were performed to detect the differences in the primary structure between the two subunits.Furthermore, the Sepharose-mercurial column was used to purify the tryptic peptide containing the essential thiol group of lombricine kinase. By this process, it was possible to obtain, in one step, the peptide in a pure state.It is interesting to point out the applicability of this method for the purification of peptides or proteins containing essential thiol groups which can be specifically labelled by reversible inhibitors.Among the dimeric guanidinophosphokinases [ 13 actually known and possessing a molecular weight of 80000 [1, 21, lombricine kinase obtained from Lumbricus terrestris muscles (Bassin parisien, France) is the only one which appears to have non-identical subunits. OnIy one binding site for the nucleotide substrate ADP-Mg and only one essential thiol group could be determined per mole of enzyme [2,3]. This raises the question of whether the chemical structures within the two subunits of lombricine kinase are identical or not, or whether a conformational change which occurs either on binding the first mole of ADPMg or one mole of -SH inhibitor on one subunit allows the other to become mireactive.The present work was undertaken to separate and isolate the two polypeptide chains of lombricine kinase by means of chromatography on Hg-coupled Sepharose.Abbreviations. Dansyl or Dns, 5-dimethylaminonaphthalene-1-sulphonyl; N,ph-F, 2,4-dinitroflnorobenzene; N,ph, 2,4-dinitrophenyI; Nbs,, 5,5'-dithio-bis(2-nitrobenzoic acid).Enzymes. Creatine kinase (EC 2.7.3.2) ; lombricine kinase (EC 2.7.3.5).This technique was made possible by the use of a. reversible -SH inhibitor : 2,4-dinitrofluorobenzene ; this reagent was specific for the essential thiol group of lombricine kinase and allowed its protection during the alkylation of the other thiol groups.After thiolysis by mercaptoethanol [4] the enzyme recovers its free essential -SH group and can therefore be bound to the Sepharose-mercurial.I n addition, this technique has been applied to purify, in one step, the tryptic peptide containing the essential thiol of lombricine kinase. EXPERIMENTAL PROCEDURE MaterialsCarboxypeptidases A and B were purchased from Worthington Biochemical Corp., trypsin (treated with diphenylcarbamyl chloride) from Seravac. Iodoacetic acid (BDH) was recrystallized in ethyl etherlpetroleum ether until...

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Spectrophotometric investigations of the interaction of native and chemically modified ATP:guanidinophosphotransferases with their substrates

Cited by 45 publications

References 25 publications

Effects of anions on a monomeric and a dimeric arginine kinase

Effects of anions on a monomeric and a dimeric arginine kinase

Structural Properties of the Creatine‐Kinase Active Site Studied by Chromophoric‐Reagent Labelling

Separation of the Two Non‐Identical Subunits of Lombricine Kinase from Lumbricus terrestris Muscle by Chromatography on Sepharose‐Mercurial

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