Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis.
The reaction of two D-amino acid oxidases from the yeasts Rhodotorula gracilis and Trigonopsis variabilis with the substrates alanine and valine in their 2-1H and 2-2H forms was studied employing the stopped-flow spectrophotometric technique. The turnover numbers at infinite substrate and oxygen concentrations were: 20,700/4,250 and 1,730/360 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,150/440 and 2,500/520 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The rates of anaerobic enzyme flavin reduction were 20,100/4,000 and 1,820/350 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,470/350 and 2,460/480 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The isotope effects on enzyme reduction were 5.0 and 5.2 for Rhodotorula and 9.9 and 5.1 for Trigonopsis D-amino acid oxidases with alanine and valine, respectively. This suggests that the intrinsic isotope effect on rupture of the substrate alpha-C-H bond can be as high as 10. The rate-determining step corresponds to the enzyme reductive half-reaction in contrast to the mammalian kidney enzyme where it is the product release from oxidized enzyme (Massey, V., and Gibson, Q.H. (1964) Fed. Proc. 23, 18-29). Upon anaerobic reaction with substrate, the yeast enzymes do not form the transient long wavelength absorbing species which are characteristic of the mammalian protein. This is due only in part to rapid dissociation of iminoacid product and is ascribed to intrinsic differences between the charge-transfer complexes of reduced enzyme flavin and product of the yeast as compared to the mammalian enzyme. With the Trigonopsis enzyme the flavin radical anion appears to be strongly stabilized and can be produced quantitatively.
Pollegioni
L
L
Department of General Physiology and Biochemistry, University of Milano, Italy.
Langkau
B
B
Tischer
W
W
Ghisla
S
S
Pilone
M S
MS
D000409:Alanine / Q000378:metabolism; D000818:Animals; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D003903:Deuterium; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D003904:Mitosporic Fungi / Q000201:enzymology*; D008954:Models, Biological; D011865:Radioisotope Dilution Technique; D012248:Rhodotorula / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013552:Swine; D014633:Valine / Q000378:metabolism
D003903:Deuterium; D003605:D-Amino-Acid Oxidase; D014633:Valine; D000409:Alanine