Pollegioni L, Blodig W, Ghisla S
Fakultät für Biologie, Universität Konstanz, D-78434 Konstanz, Germany.
J Biol Chem. 1997 Feb 21;272(8):4924-34. doi: 10.1074/jbc.272.8.4924.
The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis variabilis with [alpha-1H]- and [alpha-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of approximately 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 approximately 3.9, and the solvent isotope effect approximately 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 [alpha-1H]- and [alpha-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect approximately 6. The solvent deuterium isotope effect on the apparent rate of reduction for [alpha-1H]- and [alpha-2H]phenylglycine is approximately 2.8 and approximately 5. The rates for k-2 are 4.2 and 0.9 s-1 for [alpha-1H]- and [alpha-2H]phenylglycine, respectively, and the corresponding isotope effect is approximately 4.7. The isotope effect on alpha-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 sigma+; k-2 correlates best with sigma or sigma+ while steric parameters have little influence. This is consistent with the transition state being structurally similar to the product. The Bronsted plot of DeltaG versus DeltaG0 allows the estimation of the intrinsic DeltaG0 as approximately 58 kJ.M-1. From the linear free energy correlations, the relation of DeltaG versus DeltaG0 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 alphaC-H to the oxidized flavin N(5) position, although, clearly, they cannot prove it.
已确定三角变种曲霉的D - 氨基酸氧化酶（EC 1.4.3.3）与[α - 1H] - 和[α - 2H]苯甘氨酸反应的动力学机制。Vmax对pH的依赖性与约8.1和>9.5的pKa值相符，前者归因于一种碱，该碱需去质子化才能有效催化。周转的氘同位素效应约为3.9，溶剂同位素效应约为1.6。还原半反应是双相的，第一个快速相k2，对应底物脱氢/酶黄素还原，第二个对应产物转化/释放。酶黄素还原包括达到平衡的过程，涉及k - 2（k2的逆向反应）的有限速率。对于[α - 1H] - 和[α - 2H]苯甘氨酸，k2分别为28.8和4.6 s-1，产生约6的一级氘同位素效应。[α - 1H] - 和[α - 2H]苯甘氨酸还原表观速率的溶剂氘同位素效应分别约为2.8和约5。对于[α - 1H] - 和[α - 2H]苯甘氨酸，k - 2的速率分别为4.2和0.9 s-1，相应的同位素效应约为4.7。α - H上的同位素效应和溶剂同位素效应因此表现为相乘关系，与高度协同的过程和对称的过渡态一致。已确定携带对位取代基F、Cl、Br、CH3、OH、NO2和OCH3的苯甘氨酸的k2和k - 2值。k2与取代基体积VM和σ+呈线性相关；k - 2与σ或σ+相关性最佳，而空间参数影响较小。这与过渡态在结构上与产物相似一致。ΔG对ΔG0的布朗斯特图允许估计内在ΔG0约为58 kJ·M-1。从线性自由能相关性、ΔG对ΔG0的关系以及根据马库斯理论得出结论，过渡态中几乎没有电荷发展。这与最近解析的猪肾D - 氨基酸氧化酶的三维结构（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）一起，反对其经典形式的碳负离子机制。我们的数据与从底物αC - H向氧化黄素N(5)位置转移氢化物相符，尽管显然它们不能证明这一点。
