Marco Signorini

Inhibition of erythrocyte transglutaminase by GTP.

The guanine nucleotides GTP, GDP and GMP inhibit the activity of erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) in a decreasing order of effectiveness. The inhibition is more apparent at low than at saturating levels of calcium ions and is not due to the chelation of Ca2+, but to an interference with the process of activation by the cation. This inhibition is likely to contribute to the latency of erythrocyte transglutaminase in physiological conditions.

Identification of Transglutaminase Activity in the Leaves of Silver Beet (Beta vulgaris L.)

Summary Leaves of silver beet (Beta vulgaris L.) were shown to contain enzymes which catalyse the incorporation of primary amines into endogenous and exogenous proteins. Upon acid hydrolysis of proteins labelled with 14C-putrescine, almost all the radioactivity was recovered as the original amine. A considerable fraction of the label was present as glutaminyl-putrescine derivative in isopeptide bond after exhaustive degradation of labelled proteins with proteolytic enzymes. These results suggest the presence of transglutaminases in plant tissues. This conclusion was supported by the demonstration that the reaction was stimulated by calcium ions, although not absolutely dependent on the cation, and that it was inhibited by recognised transglutaminase substrates and by ammonium ions. The enzymes were found to be associated with the cell particulate fraction and were probably intrinsic membrane proteins because detergents were required for solubilisation of the activity. Peptides of apparent molecular mass of 65,000 daltons by SDS-PAGE were identified as organelle associated endogenous substrates.

Identification of the chemical groups involved in the binding of periodate-oxidized NADP+ to 6-phosphogluconate dehydrogenase.

Periodate-oxidized NADP+ binds specifically and reversibly to the NADP+ binding site of 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Candida utilis. The inhibition can be stabilized by reduction with sodium borohydride. It has been shown that an aldehydic group of the inhibitor forms a Schiff base with a lysine residue of the enzyme.

Evidences for the involvement of a histidine residue in the binding of the substrate to the 6-phosphogluconate dehydrogenase

Abstract The change, induced by a chemical modification, of the p K value of amino acid-ionizable groups has been used as a tool to investigate the role of these residues in the mechanism of action of enzymes. The specific ethoxyformylation of one histidine residue per subunit of 6-phosphogluconate dehydrogenase results in the reversible inactivation of the enzyme. The inactivation could be correlated to the strong decrease of the affinity of the enzyme for the substrate but not for the coenzyme. Kinetic and equilibrium dialysis experiments have indicated that the decrease, upon ethoxyformylation, of the p K value of the histidine residue is correlated to the decrease of the p K value of the group involved in the binding of the 6-phosphogluconate to the enzyme. This correlation suggests that the ionization of a histidine residue is involved in the binding of the substrate to the enzyme. The involvement of the ionization of a tyrosine residue in the binding of the coenzyme and of two other amino acid residues, with p K values of 7.2 and 8.2, in the enzyme catalysis, is also postulated.

The active site of 6-phosphogluconate dehydrogenase: A phosphate binding site and its surroundings

Abstract Tetrahedral anions bind to a phosphate binding site of 6-phosphogluconate dehydrogenase from Candida utilis , inhibit the enzyme competitively with the 6-phosphogluconate, decrease the reactivity of the SH groups, and mimic the protective effect of 6-phosphogluconate against some inactivating agents. The reaction of the enzyme with butanedione results in the inactivation of the enzyme associated with the modification of a single arginine residue per subunit. This arginine residue may be involved in the binding of the phosphate to the enzyme. Inactivation of the enzyme, upon reaction with permanganate, appears to be due to the oxidation to cysteic acid of a single cysteine residue per enzyme subunit. The reaction of the enzyme with either periodate or hexachloroplatinate causes the loss of the catalytic activity. This inactivation, due to an affinity labeling, is correlated with the oxidation of two SH groups per subunit to an S-S bridge. Photoinactivation of the enzyme by pyridoxal 5′-phosphate is also restricted to the active site of the enzyme. The lysine and the histidine residues involved in this photoinactivation should thus be in the vicinity of the phosphate binding site.

Evidence for a tyrosine residue at the triphosphopyridine nucleotide-binding site of 6-phosphogluconate dehydrogenase.

Abstract Treatment of the 6-phosphogluconate dehydrogenase from Candida utilis with tetranitromethane results in the partial inactivation of the enzyme. The nitration of approximately one tyrosine residue per enzyme subunit accounts for the loss of 70% of the enzymatic activity. The reduction of the nitrotyrosyl to aminotyrosyl residue does not induce a recovery of activity. The pH dependency of the affinity for 6-phosphogluconate and TPN of the untreated, nitrotyrosyl, and aminotyrosyl enzyme has been investigated. The nitration of one tyrosyl residue per enzyme subunit modifies the influence of the pH of the assay mixture on the affinity of the enzyme for TPN but not for 6-phosphogluconate. The reduction of the nitrotyrosyl to the aminotyrosyl enzyme reverses this effect of the nitration. These results seem to indicate that the tyrosine residue involved in the nitration is at the TPN-binding site and that its ionization, or other events connected with its nitration or amination, can influence the binding of the coenzyme to the 6-phosphogluconate dehydrogenase.

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

Abstract The reaction of NADP + with periodate yields a coenzyme analog that can be bound to the NADP + binding site of 6-phosphogluconate dehydrogenase from Candida utilis . This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.

A role for the pyridine nitrogen of reduced triphosphopyridine‐nucleotide in the mechanism of action of isocitrate dehydrogenase

The isocitrate dehydrogenase (L,-isocitrate: NADP oxidoreductase, decarboxylating, EC 1.1.1.42) catalyses, in the presence of TPN, the oxidative decarboxylation of isocitrate to a-ketoglutarate and CO,. The enzyme catalyses also a tritium exchange reaction between tritiated a-ketoglutarate and water; this reaction requires the presence of 1,4 TPNH [l] , but the role of the reduced coenzyme in this reaction is unknown. Alsot the tritium exchange reaction, catalysed by 6phosphogluconate dehydrogenase, between tritiated ribulose Sphosphate and water, requires the presence of I,4 TPNH [2] . In this case there are indications that the nitrogen of the nicotinamide ring of the reduced coenzyme influences the ionization of amino acid residues involved in both the tritium exchange reaction and in the binding of the coenzyme to the enzyme [3,4] . In the present paper we report some experiments which indicate that also in the case of isocitrate dehydrogenase the pyridine nitrogen of the reduced coenzyme seems to play a role in the tritium exchange reaction.

Differentiation between the structural and redox roles of TPNH in 6-phosphogluconate dehydrogenase

Abstract In the tritium exchange reaction catalysed by 6-phosphogluconate dehydrogenase from Candida utilis , the natural reduced coenzyme, 1–4 TPNH, has been substituted efficiently by its non enzymatically oxidizable isomer, 1–4 TPNH. This indicates that, in the tritium exchange reaction, the TPNH has not a redox but a structural role. We suggest that, as for 6-phosphogluconate dehydrogenase, isomers of the natural coenzymes may be used to differentiate, and study separately, the redox and structural roles of pyridine coenzymes in dehydrogenases.

Evidence for the proximity of two sulfhydryl groups at the active site of 6-phosphogluconate dehydrogenase☆

Abstract The reaction between 6-phosphogluconate dehydrogenase from Candida utilis and 5,5′-dithiobis(2-nitrobenzoate) results in the inactivation of the enzyme. At pH 6.0 the inactivation can be correlated with the modification of only one SH group per enzyme subunit. The modified SH group can react with another SH group forming an intramolecular disulfide bridge. Since the modified enzymes, either with an SH group modified or with a cystine disulfide bridge, are still able to bind the substrate and the coenzyme, gross conformational changes seem unlikely to have occurred. The results obtained suggest that the SH groups of two cysteine residues are located close to each other in the three-dimensional structure of the active site of the enzyme.