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 va line in their 2-H-1 and 2-H-2 forms was studied employing the stopped- flow spectrophotometric technique. The turnover numbers at infinite su bstrate and oxygen concentrations were: 20,700/4,250 and 1,730/360 ([2 -H-1]/[2-H-2]alanine and valine, respectively) for the Rhodotorula and 3,150/440 and 2,500/520 ([2-H-1]/[2-H-2]alanine and valine, respectiv ely) for the Trigonopsis enzymes. The rates of anaerobic enzyme flavin reduction were 20,100/4,000 and 1,820/350 ([2-H-1]/[2-H-2]alanine and valine, respectively) for the Rhodotorula and 3,470/350 and 2,460/480 ([2-H-1]/[2-H-2]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 wit h alanine and valine, respectively. This suggests that the intrinsic i sotope effect on rupture of the substrate alpha-C-H bond can be as hig h 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 absorbin g species which are characteristic of the mammalian protein. This is d ue only in part to rapid dissociation of iminoacid product and is ascr ibed to intrinsic differences between the charge-transfer complexes of reduced enzyme flavin and product of the yeast as compared to the mam malian enzyme. With the Trigonopsis enzyme the flavin radical anion ap pears to be strongly stabilized and can be produced quantitatively.