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The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis variabilis with [α-1H]- and [α-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of ≈8.1 and >9.5, the former of which is attributed to a base which should be deprotonated for efficient catalysis. The deuterium isotope effect on turnover is ≈3.9, and the solvent isotope effect ≈1.6. The reductive half-reaction is biphasic, the first, fast phase, k2, corresponding to substrate dehydrogenation/enzyme flavin reduction and the second to conversion/release of product. Enzyme flavin reduction consists in an approach to equilibrium involving a finite rate for k-2, the reversal of k2. k2 is 28.8 and 4.6 s-1 for [α-1H]- and [α-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect ≈6. The solvent deuterium isotope effect on the apparent rate of reduction for [α-1H]- and [α-2H]phenylglycine is ≈2.8 and ≈5. The rates for k-2 are 4.2 and 0.9 s-1 for [α-1H]- and [α-2H]phenylglycine, respectively, and the corresponding isotope effect is approx 4.7. The isotope effect on α-H and the solvent one thus behave multiplicatively consistent with a highly concerted process and a symmetric transition state.
The k2 and k-2 values for phenylglycines carrying the para substituents F, Cl, Br, CH3, OH, NO2 and OCH3 have been determined. There is a linear correlation of k2 with the substituent volume VM and with σ+; k-2 correlates best with σ or σ+ while steric parameters have little influence. This is consistent with the transition state being structurally similar to the product. The Brønsted plot of ΔGDagger versus ΔG0 allows the estimation of the intrinsic ΔG0Dagger as ≈58 kJ·M-1. From the linear free energy correlations, the relation of ΔGDagger versus ΔG0 and according to the theory of Marcus it is concluded that there is little if any development of charge in the transition state. This, together with the recently solved three-dimensional structure of D-amino acid oxidase from pig kidney (Mattevi, A., Vanoni, M.A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., and Curti, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7496-7501), argues against a carbanion mechanism in its classical formulation. Our data are compatible with transfer of a hydride from the substrate αC-H to the oxidized flavin N(5) position, although, clearly, they cannot prove it.

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 α-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.

D-Amino acid oxidase (EC 1.4.3.3) from Rhodotorula gracilis has been reconstituted with 8-chloro-, 8-mercapto-, 6-hydroxy-, 2-thio-, 5-deaza- and 1-deaza-FAD, and the properties of the resulting complexes have been studied and compared with those of the correspondingly modified pig kidney D-amino acid oxidases. Binding appears to be tight for most analogues, at least as tight as for native FAD (~10(-8) M). 8-Mercapto- and 6-hydroxy-FAD bind in their para- and ortho-quinoid forms respectively to yeast D-amino acid oxidase, inferring the presence of a positive charge near the flavin N(1) position, as in the case of the mammalian enzyme. On the other hand, important differences in active-site microenvironment emerge: solvent accessibility to flavin position 8 is drastically restricted in yeast D-amino acid oxidase as indicated by the unreactivity of 8-chloro- and 8-mercapto-FAD enzyme with thiolates and alkylating agents. Significantly different microenvironments are also likely to occur around the flavin positions N(1)-C(2) = 0, N(3)-H and N(5). This is deduced from the differences in interaction of the two proteins with 1-deaza-FAD, 5-deaza-FAD and 2-thio-FAD and from the properties of the respective complexes. The same re-side flavin stereospecificity as shown by the mammalian enzyme was determined for the yeast enzyme using 8-hydroxy-5-deaza-FAD. Thus we can deduce the presence of a similar pattern of functional groups at the active centres of the two enzymes, while the fine tuning of specificity and regulation correlate with environmental differences at specific flavin loci.