ABSTRACT
Glutamine(asparagine)ase catalyses desamidation of both L-glutamine and L-asparagine, and their D-isomers. In this study the two-pharmacophore models of main enzyme substrates and their hydrolysed analogues were design. The received models reflect two stage of substrate interaction with the enzyme active site. These models allow to explain the wide substrate specificity of glutamine(asparagine)ase.
Subject(s)
Amidohydrolases/metabolism , Computer Simulation , Drug Design , Binding Sites , Catalysis , Substrate SpecificityABSTRACT
The review summarizes and analyzes experimental evidence for the properties of glutamine(asparagine)ase from Pseudomonas aurantiaca-548. The enzyme is a tetramer having a molecular weight of 148 kD and consisting of 4 identical subunits having a molecular weight of 37 kD. For glutaminase activity, the optimum pH is in the range of 6.0-8.0, asparaginase activity increases as pH rises. The enzyme is maximally stable at pH 6.8-8.0. The Michaelis constants are 5.3 +/- 0.7 x 10(-6) M for L-glutamine and 5.7 +/- 0.1 x 10(-6) M for asparagine. The reaction products L-aspartate and L-glutamate are competitive inhibitors anazaserine and 6-diase-5-oxo-1-norleucine are classic inhibitors of glutamine(asparagine)ase. The review also presents data on the conditions for culturing Ps. aurantiaca, on the procedures for isolating glutamine(asparagine)ase from biomass of this microbe, on substrate specificity. The results of searching for regulators of catalytic activity, as well as agents enhancing the resistance of enzymes to heat exposures are considered in the paper. Whether the properties of glutamine(asparagine)ase are in conformity with the criteria for primary choice of promising antitumor agents is discussed.
Subject(s)
Amidohydrolases , Pseudomonas aeruginosa/enzymology , Amidohydrolases/isolation & purification , Amidohydrolases/metabolism , Asparagine/metabolism , Catalysis , Glutamine/metabolism , Hot Temperature , Molecular Weight , Pseudomonas aeruginosa/growth & development , Substrate SpecificityABSTRACT
The effect of two reagents on glutamin (asparagin) ase from Pseudomonas aurantiaca-548 has been studied. 2,3-butanedione which modified arginine residues was ineffective for the inactivation of the enzyme. The enzyme was completely inactivated in the presence of N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K). The effects of pH, reagent concentration, competitive inhibitors and their analogues on the rate or degree of enzyme inactivation were studied. The experimental results suggest that the carboxyl groups localized at the active site of glutamin (asparagin) ase are probably essential for the substrate binding.
Subject(s)
Amidohydrolases , Antineoplastic Agents , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/isolation & purification , Enzyme Inhibitors/pharmacology , Epoxy Compounds , Indicators and Reagents , Isoxazoles , Pseudomonas/enzymologyABSTRACT
Incubation of homogeneous glutamin(asparagin)ase from Pseudomonas aurantiaca with 6-diazo-5-oxo-L-norleucine (DON) and azaserine leads to an almost complete inactivation of the enzyme. The inactivation process in both cases involves the step of reversible binding of the enzyme with the inhibitor into a complex and subsequent modification of the enzyme within this complex. The data on saturation of the enzyme by low concentrations of inhibitors, the protective effect of substrate and its analogs as well as of the competitive inhibitor and product of the enzymatic reaction, L-aspartate, suggest that the modification of functional groups takes place in the enzyme active site. The presence of essential threonine hydroxyl groups in/or near the enzyme active site is surmised.
Subject(s)
Affinity Labels , Amidohydrolases/antagonists & inhibitors , Azaserine/pharmacology , Azo Compounds/pharmacology , Diazooxonorleucine/pharmacology , Pseudomonas/enzymology , Amino Acids/pharmacology , Hydrogen-Ion Concentration , KineticsABSTRACT
The effect of substrate analogues on glutamin-(asparagin-)ase from Pseudomonas aurantiaca-548 has been studied. The enzyme was demonstrated to be highly sensitive to the the action of 6-diazo-5-oxo-L-norleucine and azaserine. L-isomers of glutamine, aspartate, glutamate and several other substrate analogues with free alpha-amino groups protected the enzyme against the inhibitory DON effect. Thus, thorough preliminary selection of appropriate inhibitors, their dosage and treatment duration is needed for the recommendation of combined enzyme-inhibitor application in anti-tumour chemotherapy.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Azaserine/pharmacology , Azo Compounds/pharmacology , Diazooxonorleucine/pharmacology , Antineoplastic Agents , Pseudomonas/drug effects , Pseudomonas/enzymology , Substrate Specificity/drug effectsABSTRACT
In studies on kinetics of thermoinactivation of glutaminase (asparaginase) from Ps. arantiaca BKMB-548 at 50 degrees and pH 7.0 in presence or in absence of L-glutamate the enzyme inactivation was found to obey the first order equation. Both the glutaminase and asparaginase activities decreased at a similar rate. L-Glutamate stabilized the enzyme due to direct interaction with its molecule. Stability of the complex formed was evaluated quantitatively. L-Glutamate reacted apparently with a specific site on the surface of the enzyme molecule; Kdiss was 0.42 +/- 0.03 mM at pH 7.0 and 50 degrees. No cooperative effect was found. L-Aspartate protected the enzyme completely; stabilizing effects of L-cysteine, L-serine and glycine were similar to the effect of L-glutamate (94%, 84%, 83% and 82%, respectively). At the same time, glutarate, succinate, alpha-ketobutyrate, alpha-ketoglutarate, gamma-aminobutyrate and N-benzoyl glutamate did not exhibit the stabilization effect. The data obtained suggest that the high stabilizing effect might exhibit only the substances containing simultaneously free alpha-NH2 and alpha-COOH groups in a molecule, whereas presence of COOH groups at beta--or gamma-carbon atoms was not essential for the stabilizing effect.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Hot Temperature , Pseudomonas/enzymology , Binding Sites , Chemical Phenomena , Chemistry , Kinetics , Sodium Glutamate/pharmacology , Substrate SpecificityABSTRACT
A method for isolation of glutamin (asparagin) ase from Pseudomonas aurantiaca BKMB-548 has been developed. The enzyme preparation is homogeneous during polyacrylamide gel electrophoresis (pH 7.5 and 7.0) with SDS. The pH-optima of the enzyme thermal stability and of the glutaminase activity are equal to 6.0-8.0. At higher pH values the asparaginase activity increases within the pH range of 4-9. The amino acid composition of glutamin(asparagin)ase has been determined.
