Effect of hydrogen peroxide on d-amino acid oxidase from Rhodotorula gracilis

SummaryEchis colorata bites cause impairment of platelet aggregation and hemostatic disorders. The mechanism by which the snake venom inhibits platelet aggregation was studied. Upon fractionation, aggregation impairment activity and L-amino acid oxidase activity were similarly separated from the crude venom, unlike other venom enzymes. Preparations of L-amino acid oxidase from E.colorata and from Crotalus adamanteus replaced effectively the crude E.colorata venom in impairment of platelet aggregation. Furthermore, different treatments known to inhibit L-amino acid oxidase reduced in parallel the oxidase activity and the impairment potency of both the venom and the enzyme preparation. H2O2 mimicked characteristically the impairment effects of L-amino acid oxidase and the venom. Catalase completely abolished the impairment effects of the enzyme and the venom. It is concluded that hydrogen peroxide formed by the venom L-amino acid oxidase plays a role in affecting platelet aggregation and thus could contribute to the extended bleeding typical to persons bitten by E.colorata.

Download Full-text

FUNCTIONAL AND STRUCTURAL ASPECTS OF D-AMINO ACID OXIDASE FROM Rhodotorula gracilis PROBED BY LIMITED PROTEOLYSIS

Flavins and Flavoproteins 1993 ◽

10.1515/9783110885774-032 ◽

1994 ◽

pp. 171-174

Keyword(s):

Amino Acid ◽

Limited Proteolysis ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Rhodotorula Gracilis ◽

Structural Aspects

Download Full-text

Nitroalkanes as Reductive Substrates for Flavoprotein Oxidases

Zeitschrift für Naturforschung B ◽

10.1515/znb-1972-0914 ◽

1972 ◽

Vol 27 (9) ◽

pp. 1052-1053 ◽

Cited By ~ 16

Author(s):

David J. T. Porter ◽

Judith G. Voet ◽

Harold J. Bright

Keyword(s):

Hydrogen Peroxide ◽

Amino Acid ◽

Glucose Oxidase ◽

Ph Dependence ◽

Kinetic Mechanism ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Kinetics Of ◽

Oxidase Reaction ◽

Detailed Kinetic Mechanism

Nitroalkanes have been found to be general reductive substrates for D-amino acid oxidase, glucose oxidase and L-amino acid oxidase. These enzymes show different specificities for the structure of the nitroalkane substrate.The stoichiometry of the D-amino acid oxidase reaction is straightforward, consisting of the production of one mole each of aldehyde, nitrite and hydrogen peroxide for each mole of nitroalkane and oxygen consumed. The stoichiometry of the glucose oxidase reaction is more complex in that less than one mole of hydrogen peroxide and nitrite is produced and nitrate and traces of 1-dinitroalkane are formed.The kinetics of nitroalkane oxidation show that the nitroalkane anion is much more reactive in reducing the flavin than is the neutral substrate. The pH dependence of flavin reduction strongly suggests that proton abstraction is a necessary event in catalysis. A detailed kinetic mechanism is presented for the oxidation of nitroethane by glucose.It has been possible to trap a form of modified flavin in the reaction of D-amino acid oxidase with nitromethane from which oxidized FAD can be regenerated in aqueous solution in the presence of oxygen.

Download Full-text

Substitution of the critical methionine residues in Trigonopsis variabilis ?-amino acid oxidase with leucine enhances its resistance to hydrogen peroxide

FEMS Microbiology Letters ◽

10.1016/s0378-1097(00)00151-8 ◽

2000 ◽

Vol 186 (2) ◽

pp. 215-219 ◽

Cited By ~ 1

Author(s):

S Ju

Keyword(s):

Hydrogen Peroxide ◽

Amino Acid ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Trigonopsis Variabilis ◽

Methionine Residues

Download Full-text

The formation of choleglobin and the role of catalase in the erythrocyte

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1949.0035 ◽

1949 ◽

Vol 136 (884) ◽

pp. 435-448 ◽

Cited By ~ 11

Keyword(s):

Hydrogen Peroxide ◽

Ascorbic Acid ◽

Amino Acid ◽

Catalase Activity ◽

Acid Oxidase ◽

Small Decrease ◽

Amino Acid Oxidase ◽

Oxidase System ◽

Inverse Proportion

In haemolysates of non-nucleated erythrocytes there is an inverse proportion between catalase activity and rate of choleglobin formation on addition of ascorbic acid. In the intact erythrocytes catalase protects haemoglobin against oxidation and further destruction by the hydrogen peroxide generated by the D-amino-acid oxidase system or by physiological concentrations of ascorbic acid and glutathione. Acid destromatization of haemolyzed horse erythrocytes causes a small decrease in the catalase activity and an increased rate of inactivation of the remaining catalase by ascorbic acid. The liberation of copper from haemocuprein is quantitatively insufficient to explain the decreased stability of the catalase. Exposing duck oxyhaemoglobin, but not reduced haemoglobin, to a pH of 5⋅5 to 5⋅8, causes an alteration which is apparent from the increase of the rate of choleglobin formation. The mechanism of this alteration is discussed. It partly explains the 'stroma effect', at least in duck erythrocytes. In addition, in the latter, there is a true stroma effect. Choleglobin formation in the presence of ascorbic acid is accelerated by a variety of substances. Some of these perturb haemoglobin, while others increase the formation of hydrogen peroxide from ascorbic acid. The implications of our findings on the mechanism of choleglobin formation and on the role of catalase in the erythrocyte are discussed.

Download Full-text

Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters

Biochemical Journal ◽

10.1042/bj3300311 ◽

1998 ◽

Vol 330 (1) ◽

pp. 311-314 ◽

Cited By ~ 3

Author(s):

F. RAMÓN ◽

M. P. CASTILLÓN ◽

I. DE LA MATA ◽

C. ACEBAL

Keyword(s):

Amino Acid ◽

Kinetic Parameters ◽

Ph Dependence ◽

Competitive Inhibitor ◽

Chemical Mechanism ◽

Acid Oxidase ◽

Protonation State ◽

Amino Acid Oxidase ◽

Basic Group ◽

Rhodotorula Gracilis

The variation of kinetic parameters of D-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. D-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the β-carboxylic group of the inhibitor.

Download Full-text