Comparative Structural Studies of the Active Site of ATP: Guanidine Phosphotransferases

der, Terrossian E; Desvages, Giséle; La, Pradel; Kassab, Ridha; Nguyen-Van-Thoai,

doi:10.1111/j.1432-1033.1971.tb01581.x

Cited by 17 publications

(3 citation statements)

References 20 publications

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“…I n the case of ATP : guanidine phosphotransferases deriving from several origins [22] which are currently being studied in our laboratory, an extensive work has been made giving chemical and physical evidence of the homology of their active sites [ 10, 27,28] in which cysteine and lysine have been found to be implicated in the binding of the guanidine [l, 101 and nucleotide substrates, respectively [l, lo], whereas histidine was essential for the phosphoryl group transfer [l, 101. This data was obtained by means of chemical methods employing selective reagents [e.g.…”

Section: Effect Of Tyrosyl Modification On the Conformation Of Arqwinmentioning

confidence: 99%

Effects of Iodination and Acetylation of Tyrosyl Residues on the Activity and Structure of Arginine Kinase from Lobster Muscle

Fattoum

Kassab

Pradel

1971

European Journal of Biochemistry

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The iodination and 0-acetylation of tyrosyl residues in arginine kinase from lobster muscle (Homarus vulgaris) with iodine and N-acetylimidazole have been studied after reversible blocking of the thiol groups.The conversion of one tyrosyl residue to monoiodotyrosine, as measured by spectrotitration and by radioactive iodine labeling, resulted in a total loss of activity.The extent of the conformational changes occurring after nitration by tetranitromethane and iodination was studied by the optical rotary dispersion and the hydrogen-tritium exchange techniques.The functional properties of this tyrosyl residue were underlined by the inhibition of arginine kinase after o-acetylation and the ability of the modified protein to recover full activity upon tyrosyl de-o-acetylation with neutral hydroxylamine.The present investigation was initiated in an effort to collect more information about the importance of aromatic side chains in the catalytic activity and the structure of arginine kinase from lobster muscle. The tyrosyl residues have focused our special attention since we have found [l] that the interaction of this phosphagen kinase with its guanidine substrates as well as with some hydrophobic L-a-amino acids or specific -SH reagents could be visualized spectrally by the occurrence of a characteristic difference spectrum involving a blue shift of tyrosine a t 280 and 287nm and a hypochromic phase a t 239 nm; this latter spectral modification was shown t o be a result of the direct participation of the essential cysteinyl residue of the protein in the binding process [2]. This spectral data pointed out the possible proximity of functional sulfhydryl and tyrosyl groups in the enzyme active site.I n an earlier report [3] we studied the inactivation of arginine kinase by nitration of a single tyrosyl residue with the specific tyrosine reagent, tetranitromethane. On the basis of the results obtained concerning the immunochemical reactivity and optical rotary dispersion properties of the mononitrated protein, we concluded that a gross conformational change accompanied the complete inhibition of the enzyme observed. To extend and confirm these observations, we have examined, during the course of this study, the effect of the chemical modification of the tyrosyl residues in arginine b a s e with two other reagents : iodine and N-acetylimidazole. It Enzyme. Arginine kinase (EC 2.7.3.3).30 Eur. J. Bioehem., Vol. 22 has been shown that monoiodination of a single tyrosyl residue altered both the catalytic activity and the three-dimensional structure of the enzyme ; the importance of this critical tyrosyl side chain in the maintenance of the native conformation of arginine kinase was further illustrated by the reversible inhibition of the enzyme upon tyrosyl o-acetylation with N-acetylimidazole. A preliminary account of this work was presented [4]. EXPERIMENTAL PROCEDURE MATERIALSArginine kinase from lobster muscle Homarus vulgaris was prepared as previously described [5,6]. Its specific activity was about 240 unitslmg. Be...

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Section: Effect Of Tyrosyl Modification On the Conformation Of Arqwinmentioning

confidence: 99%

Effects of Iodination and Acetylation of Tyrosyl Residues on the Activity and Structure of Arginine Kinase from Lobster Muscle

Fattoum

Kassab

Pradel

1971

European Journal of Biochemistry

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“…Analysis of the peptide we obtained showed that it was exactly identical to the peptide previously isolated by means of more "classical" methods [17]. Furthermore this new method revealed its usefulness in several ways.…”

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

confidence: 66%

“…The protein suspension was then submitted to tryptic hydrolysis a t 37 "C with an enzyme/substrate molar ratio of 1/100 for 3 h, then of 1/50 for one night as previously described [17]. The reaction was stopped by lowering the p H to 4 with formic acid.…”

Section: Binding Of Free Essential -Sh Tryptic Peptide Of Lombricine 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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Sequence homology and structure predictions of the creatine kinase isoenzymes

Mühlebach¹,

Groß²,

Wirz³

et al. 1994

Cellular Bioenergetics: Role of Coupled Creatine Kinases

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Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guanidino kinases. A 'CK framework' is defined, consisting of the most conserved sequence blocks, and 'diagnostic boxes' are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis. (Mol Cell Biochem 133/134: 245-262, 1994).

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Comparative Structural Studies of the Active Site of ATP: Guanidine Phosphotransferases

Cited by 17 publications

References 20 publications

Effects of Iodination and Acetylation of Tyrosyl Residues on the Activity and Structure of Arginine Kinase from Lobster Muscle

Effects of Iodination and Acetylation of Tyrosyl Residues on the Activity and Structure of Arginine Kinase from Lobster Muscle

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

Sequence homology and structure predictions of the creatine kinase isoenzymes

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