Claude Roustan

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.

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.

Yeast 3-phosphoglycerate kinase Essential arginyl residues at the 3-phosphoglycerate binding site

Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate.

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.

Preliminary X-ray diffraction studies on lobster ATP: l-Arginine phosphotransferase

Abstract Lobster muscle arginine kinase crystals have been grown by a procedure of gaseous diffusion. The resulting crystals are monoclinic. space group P21 with unit cell dimensions of a = 75.9 A , b = 88.3 A , c = 58.2 A , β = 102.0 ° . Considerations of cell volume and protein molecular weight indicated the presence of two molecules in the asymmetric unit. A search for heavy-atom derivatives is in progress.

The tyrosyl residues of yeast 3-phosphoglycerate kinase: Reactivity toward iodine

Recent X-ray crystallography data [ 1,2] suggest that conformational changes observed in 3-phosphoglycerate kinase are associated with the interaction of this enzyme with its substrates. These conformational changes could be involved in the various stages of catalysis and this could explain kinetic results [3] . The interesting feature of the yeast enzyme is that it possesses only one cysteine residue which is not at the active center [ 1,4,5]. On the basis of results obtained previously [6,7] a carboxyl residue is implicated in the transphosphorylation process. Furthermore, the carbethoxylation of histidine residue is found to induce a loss of activity concomitant with a weak conformational change 161. Differential spectrophotometric investigations of the interaction of 3-phosphoglycerate with phosphoglycerate kinase have demonstrated that the binding of the acceptor produces the perturbation of a phenolic chromophore in the enzyme [8]. Since we have found, as mentioned in this paper, that all chromophoric residues are buried, tyrosyl residues have focused our special attention. Herein, we report some preliminary results obtained by chemical modification of essential tyrosyl residues in yeast 3-phosphoglycerate kinase.

Essential carboxyl groups in yeast hexokinase

Although a great deal of kinetic data are available on yeast hexokinase and several schemes have been proposed, the mechanism of its action is far from being understood [ 1,2] . It is generally agreed, however, that the transphosphorylation step requires the formation of a ternary complex between the enzyme and its substrates, ATP-Mg and D-glucose. Although the pH dependence of the enzyme activity suggests that at least one ionisable group, such as the imidazole of histidine or the y-carboxyl group of aspartic or glutamic acids, takes part in the catalytic process [3, 41, the nature of the amino acid residues involved in enzymatic catalysis or substrate binding remains to be determined. According to the results of Grouselle et al. [5], as well as our own unpublished results, the histidine residues do not appear to be associated either with the catalytic step or with the enzyme-substrate interactions. Thus, the inactivation observed when the enzyme is carboethoxylated is caused by a conformational change in the enzyme protein rather than by the modification of an essential histidine residue. It seemed, therefore, important to investigate the effect of specific carboxyl group reagents upon the enzyme activity. Water soluble carbodiimides have been shown to be good reagents for this purpose [6]. This paper reports some preliminary results obtained by chemical modification of essential carboxyl groups in yeast hexokinase.