Ridha Kassab

Site actif des ATP: Guanidine phosphotransférases: II. Mise en évidence de résidus histidine essentiels au moyen du pyrocarbonate d'éthyle

Abstract The active site of ATP: guanidine phosphotransferases. II. Evidence for a critical histidine residue through use of a specific reagent, diethylpyrocarbonate 1. 1. ATP-creatine phosphotransferase (EC 2.7.3.2) and ATP: l -arginine phosphotransferase (EC 2.7.3.3) are inhibited at pH 6.1 by diethylpyrocarbonate, their reactive sulfhydryl groups being reversibly masked or not. The extent of inactivation is first-order with respect to time and inhibitor concentration. The ability of the substrates, used separately or mixed, to protect arginine kinase against inhibition, suggests the involvement of the modified group in the enzymic reaction. Nevertheless, in the case of creatine kinase no protection has been observed. 2. 2. The carbethoxylated enzymes develop a difference ultraviolet spectrum with a maximum peak at 240 mμ, a specific feature of N -carbethoxyimidazole formation. From the kinetic data of spectrophotometric titrations and loss of activity, it is concluded that one histidine residue and two histidine residues are modified per mole of arginine kinase and creatine kinase respectively, causing total inactivation. 3. 3. Advantages of diethylpyrocarbonate as a protein-histidyl reagent are demonstrated by the specific titration of the histidine content in both denaturated enzymes. Furthermore it is shown that the number and reactivity of -SH groups are not modified in the carbethoxylated inhibited phosphokinases.

Site actif des ATP: Guanidine phosphotransférases: I. Réaction des groupes ε-NH2 lysine essentiels avec le i-diméthylaminonaphtalène-5-sulfochlorure

Abstract The active site of ATP: guanidine phosphotransferases. I. Reaction of the essential e-NH2 lysine groups with i -dimethylaminonaphthalene-5-sulphonylchloride 1. 1. ATP: creatine phosphotransferase (EC 2.7.3.2) and ATP: l -arginine phosphotransferase (EC 2.7.3.3) are specifically inhibited by i -dimethylaminonaphthalene-5-sulfonylchloride, their essential sulphydryl groups being reversibly masked. The metal complexes Mg-ATP and Mg-ADP are shown to increase the inactivation rate of creatine kinase only. 2. 2. The yellow fluorescent dye-labelled enzymes exhibit a characteristic absorption spectrum with an absorption maximum at 335 mμ. The extent of inhibition is in good agreement with the different degrees of labelling, a fact which suggests that the dye has reacted at an enzymatically active site. Complete loss of activity is associated with the binding of two moles of dansyl per mole of creatine kinase and with one mole of dye per mole of arginine kinase. 3. 3. High voltage electrophoresis and thin-layer chromatography of the single fluorescent derivative isolated after pronase digestion of the dansylated enzymes led to the identification of e-DNS-lysine; these data provide evidence that the residue affected in both proteins is a lysine side chain.

Interaction des ATP:Guanidine phosphotransférases avec leurs substrats, étudiée par spectrophotometrie différentielle

Abstract Differential spectrophotometry of ATP:guanidine phosphotransferase-substrate complexes 1. 1. The difference spectra which result from the binding of nucleotide and guanidine substrates to creatine kinase and arginine kinase has been studied. p] ADP produces specific shift in the absorption spectrum towards higher wave lengths when interacting with both enzymes. The change observed with ATP is quite different and results from a smaller absorption of the adenine ring. When present, the metal Mg 2+ does not change the spectra. Spectrophotometric titrations with nucleotides indicate a stoichiometry of one mole of nucleotide per mole of arginine kinase and two moles of nucleotide per mole of creatine kinase. The dissociation constants for the nucleotide-phosphagene kinase complexes derived from spectral titration data are similar to those obtained from kinetic studies. 2. 2. The binding of creatine to creatine kinase provides no difference spectrum. But a specific one accompanies the binding of l -arginine and l -arginine phosphate to argnine kinase with tyrosyl peaks at 280 and 287 mμ and a hypochromic phase at 239 mμ. 3. 3. Identical difference spectra are also obtained with various hydrophobic l -amino acids such as isoleucine, valine, etc. , suggesting interaction between the enzyme active site and the structure of the studied derivatives. This fact is supported by the protecting effect afforded by the above amino acids against sulfhydryl reagents inhibition. On the other hand, alkylation of arginine kinase with iodoacetamide provides a difference spectrum showing the tyrosyl peaks produced with l -arginine and l -amino acids. The S -carboxylmethyl enzyme fails to bind arginine or amino acids while it still reacts with nucleotides. These experiments suggest that the process of binding l -arginine and some l -amino acids to the arginine kinase active site involves the interaction of the essential sulfhydryl group with the function of these compounds.

Composition en acides amines de l'ATP: l-arginine phosphotransferase cristallisee

Abstract Amino acid composition of crystalline ATP: l -arginine phosphotransferase 1. 1. ATP: l -arginine phosphotransferase (EC 2.7.3.3) has been crystallized from lobster muscle. Its specific activity was found to be 210–230 μmoles of P transferred per min per mg protein. 2. 2. From the amino acids analysis performed with a yield of 97.56%, the lobster enzyme was shown to have the following composition: Asp 38, Glu 46, Arg 19, Lys 31, Thr 21, Ser 20, His 8, Pro 11, Try 2, Tyr 11, Phe 20, Gly 28, Ala 28, Val 22, Ile 20, Leu 33, Met 8, Cyś 6, amide-N 23. 3. 3. The calculated molecular weight is 42 150 and the specific volume 0.731 ml/g.

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

Abstract The nucleotide dissociation constants and the number of binding sites for different phosphagen kinases have been determined. It is shown that the spectral patterns observed are similar for all the enzymes studied and may result from chromophoric perturbation of nucleotides as well as from specific alterations of the micro-environment of the protein-binding sites. The results obtained in the study of the binding of several nucleotide analogs to arginine kinase indicate that the formation of active nucleotide-enzyme complexes requires the presence of the γ- and β-phosphoryl groups as well as the 6-amino and N-1 groups in the purine ring. The spectral patterns observed with the native enzymes have been compared with those of chemically modified arginine and creatine kinases. Dansylation of the essential lysyl residues alters the binding properties of ADPMg 2+ but has no effect on ATPMg 2+ , and no enzyme-substrate complex is observed between 1-dimethyl-amino-5-naphthalenesulphonyl(DNS)-arginine kinase and l -arginine. Carbethoxylation of the active histidine residue in both enzymes causes no modification of the difference spectra though no transphosphorylation occurs. Nitration of one tyrosine residue in arginine kinase abolishes the interaction between this enzyme and its substrates, and whereas the modification of lysyl and histidyl groups does not result in any conformational changes, tyrosine nitration produces an important decrease in the Cotton effect at 233 mμ.

Comparaison des groupes SH reactifs des ATP:guanidines phosphotransferases

Comparative study of the reactive SH groups of ATP:guanidine phosphotransferases 1. 1. With 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence of urea, 6 SH groups are titrated in ATP:creatine phosphotransferase (EC 2.7.3.2), ATP:arginine phosphotransferase (EC 2.7.3.3) and ATP:lombricine phosphotransferase (EC 2.7.3.5); 14 SH groups are found in ATP:taurocyamine phosphotransferase (EC 2.7.3.4) and 16 in ATP:guanidinoacetate phosphotransferase (EC 2.7.3.1). In the absence of urea, only 1 SH group is titrated in lombricine kinase, 2 in creatine and taurocyamine kinases, and 6 in arginine and glycocyamine kinases. 2. 2. At pH 8.5, 3 of the SH groups of arginine kinase react at once with DTNB; at pH 7, only one SH is immediately titratable. The blocking of this SH group results in the complete inhibition of the enzyme. l-Arginine efficiently antagonizes the inhibitor; Mg-ATP and Mg-ADP decrease the rate of alkylation but do not restore the inactivation of arginine kinase. 3. 3. Stoichiometric inhibition by DTNB reveals the presence of one reactive SH group in arginine and lombricine kinases and of 2 essential SH groups in glycocyamine, creatine and taurocyamine kinases. Guanidine substrates, arginine and lombricine, efficiently protect their respective enzymes against inactivation by DTNB; in contrast glycocyamine and creatine do not protect their corresponding enzymes. Moreover, Mg-ATP protects glycocyamine and creatine kinases but not the arginine and lombricine kinases. Taurocyamine kinase is intermediate between these 2 groups of enzymes: it is protected from DTNB by both guanidine and nucleotide substrates. 4. 4. Fingerprints of N-[I-14C]ethylmaleimide-labelled creatine, taurocyamine and lombricine kinases show the presence of approximately the same number of peptides, 46, which are mainly neutral or basic. Arginine kinase, the molecular weight of which is half that of the other 3 enzymes, produces nearly the same number of peptides, 45–48. On autoradiograms of the fingerprints of the 4 enzymes, the main radioactivity seems to be situated in the same peptide.

Comparaison des poids moleculaires des ATP:guanidino phosphotransferases

Summary The molecular weights of four new ATP : guanidino phosphotransferases (EC Class 2.7.3) (taurocyamine kinase (EC 2.7.3.4), glycocyamine kinase, hypotauro-cyamine kinase (EC 2.7.3,6) and lombricine kinase (EC 2.7.3.5)) have been determined by gel filtration on Sephadex G-100 and density-gradient ultracentrifugation, using pure creatine- and pure arginine-kinases as standard proteins (respective molecular weights 81 000 and 43 000). The molecular weights of homogenous preparations of taurocyamine- and lombricine-kinases determined by analytical ultracentrifugation are the same as those of the partially purified preparations of the same enzymes determined by other techniques. The molecular weights of these proteins lie in the range of 80 000–87 000 (taurocyamine kinase), 83 000 (hypotaurocyamine kinase), 79 000–82 000 (glycocyamine kinase) to 74 000–77 000 (lombricine kinase).

Studies on the partial exchange and overall reactions catalyzed by native and modified arginine kinase from Homarus vulgaris muscle.

Abstract Initial velocity and partial exchange studies are performed on arginine kinase (ATP: l -arginine phosphotransferase, EC 2.7.3.3) from Homarus vulgaris muscle. The steady-state kinetic patterns suggest a reaction mechanism proceeding via interconversion of ternary complexes, but we can also observe partial exchange reactions between ATP and ADP or arginine phosphate and arginine. Properties of these exchange reactions are investigated. In addition, arginine kinase is the only phosphagen kinase studied which catalyses this partial exchange. Comparison between partial exchange rates related to native and specifically inhibited arginine kinase leads to confirmation of our results previously obtained concerning the role of several essential amino acid residues in the active site: dansylation of one lysyl residue or carboxymethylation of one cysteinyl residue prevents the formation of guanidine—enzyme complexes, thus only the arginine phosphate—arginine exchange is abolished. Carbethoxylation of the histidyl residue implicated in the catalytic process suppresses the two partial exchange reactions.

Interaction of the heavy chain of gizzard myosin heads with skeletal F-actin

To probe the molecular properties of the actin recognition site on the smooth muscle myosin heavy chain, the rigor complexes between skeletal F-actin and chicken gizzard myosin subfragments 1 (S1) were investigated by limited proteolysis and by chemical cross-linking with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide. Earlier, these approaches were used to analyze the actin site on the skeletal muscle myosin heads [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Biochemistry 20, 2110-2120; Labbé, J.P., Mornet, D., Roseau, G., & Kassab, R. (1982) Biochemistry 21, 6897-6902]. In contrast to the case of the skeletal S1, the cleavage with trypsin or papain of the sensitive COOH-terminal 50K-26K junction of the head heavy chain had no effect on the actin-stimulated Mg2+-ATPase activity of the smooth S1. Moreover, actin binding had no significant influence on the proteolysis at this site whereas it abolished the scission of the skeletal S1 heavy chain. The COOH-terminal 26K segment of the smooth papain S1 heavy chain was converted by trypsin into a 25K peptide derivative, but it remained intact in the actin-S1 complex. A single actin monomer was cross-linked with the carbodiimide reagent to the intact 97K heavy chain of the smooth papain S1. Experiments performed on the complexes between F-actin and the fragmented S1 indicated that the site of cross-linking resides within the COOH-terminal 25K fragment of the S1 heavy chain. Thus, for both the striated and smooth muscle myosins, this region appears to be in contact with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural aspects of actomyosin interaction

Actin binding to myosin-S1 modulates the limited tryptic cleavage of the COOH-terminal region of the 95K heavy chain at the joint connecting the 75K and 20K peptide units; concomitantly actin affords total protection against the resulting loss of acto-S1 Mg2+-ATPase activity. The specificity of the actin effect is illustrated by the fact that it exerts itself not only on free S1 but also on the intact myosin molecule. Mg2+-ATP and Mg2+-ADP impair the protective action of actin to an extent closely related to their respective affinity for the acto-S1 complex. Tryptic fragmentation of S1 heavy chain under highly controlled conditions, using trypsin to S1 weight ratios in the range 1:1000 - 1:1500 led us to establish that peptide bond cleavage at the 75K-20K junction is a sequential process giving rise first to a 22K peptide intermediate which is subsequently converted to the stable 20K fragment. Most importantly, it is also demonstrated that the loss of S1 activation by actin is not due to the initial scission of the 75K-22K linkage but is intimately associated with the breakdown of the 22K precursor into its 20K moiety. Three trypsin-modified S1 derivatives, the heavy chain of which is a complex of two or three fragments, were purified. A detailed analysis of the C-termini of these fragments, as compared to the C-terminal structure of the intact heavy chain, indicated that the 20K fragment is formed mainly through the degradation of a NH2-terminal 2K segment in the 22K precursor and that this proteolytic event is the only one accounting for the acto-S1 ATPase loss. Cross-linking experiments exploiting the reaction of a carbodiimide reagent with rigor complexes containing either fluorescent actin or fluorescent fragmented S1 revealed unequivocally the attachment of the actin monomer to recognition sites on the 20K and 50K units of S1 heavy chain. Specific interactions between the C-terminal 20K domain and light chain LC2 are proposed as being part of the molecular mechanism of the myosin-linked regulation of actomyosin interaction.