Specificity and kinetics of Rhodotorula gracilis D-amino acid oxidase.
D-Amino acid oxidase purified from the yeast Rhodotorula gracilis is a flavoenzyme which does not require exogenous FAD for maximum activity. The enzyme showed temperature and pH activity optima centred between 40 and 45 degrees C and between 8.0 and 8.5, respectively; a broad pH and ionic strength range of stability and a more limited range of thermostability was determined. The enzyme stability was markedly influenced by the presence of 2-mercaptoethanol. Apparent kinetic parameters for a number of substrates were determined: nonpolar and aromatic D-amino acids appeared to be the best substrates. Steady state measurements carried out at different oxygen concentrations indicated that for D-alanine the kinetic pattern is consistent with a Ping Pong Bi Bi mechanism; kcat values on D-alanine and D-valine are 43,250 min-1 and 31,370 min-1, respectively. L-Amino acids did not inhibit enzyme activity; several aromatic and aliphatic carboxylic acids proved to be competitive inhibitors of the enzyme and their ki values were determined. The reported properties of R. gracilis D-amino acid oxidase markedly distinguish it from other characterized D-amino acid oxidases.
Department of General Physiology and Biochemistry, University of Milano, Italy.
L
L
Pollegioni
A
A
Falbo
M S
MS
Pilone
D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012248:Rhodotorula / Q000201:enzymology*; D013696:Temperature
D003605:D-Amino-Acid Oxidase