Macheroux P, Kieweg V, Massey V, Söderlind E, Stenberg K, Lindqvist Y
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor.
Eur J Biochem. 1993 May 1;213(3):1047-54. doi: 10.1111/j.1432-1033.1993.tb17852.x.
The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.
对菠菜乙醇酸氧化酶（其活性位点的酪氨酸129被苯丙氨酸取代，即Y129F乙醇酸氧化酶）的酶学性质和三维结构进行了研究。突变体的结构未受干扰，这有助于对生化数据的解读。Y129F乙醇酸氧化酶的吸收光谱在364和450nm处有最大值（εmax = 11400 M-1 cm-1）。该光谱表明黄素处于其正常的质子化形式，即Y129F突变体不像野生型酶那样降低氧化型黄素N(3)的pKa[马谢鲁克斯，P.，梅西，V.，蒂勒，D. J.，和沃洛基塔，M.（1991）《生物化学》30，4612 - 4619]。Y129F乙醇酸氧化酶的pH滴定证实了这一点，结果表明pKa高于pH 9。与野生型乙醇酸氧化酶不同，草酸盐不会干扰Y129F乙醇酸氧化酶的吸收光谱。此外，草酸盐不会抑制突变酶的酶活性。野生型乙醇酸氧化酶的一些典型特征，如与黄素N(1)-C(2 = O)附近带正电荷的赖氨酸侧链相关的阴离子黄素半醌的稳定以及紧密的N(5)-亚硫酸盐加合物的形成，在Y129F突变蛋白中均得以保留。Y129F乙醇酸氧化酶表现出约为野生型活性3.5%的活性。突变体的较低周转数为0.74 s-1，而野生型酶为20 s-1，这相当于过渡态能量增加了约7.8 kJ/mol。稳态分析得出乙醇酸和氧气的Km值分别为1.5 mM和7 μM。乙醇酸的Km略高于野生型乙醇酸氧化酶的Km值（1 mM），而氧气的Km值则低得多。与野生型乙醇酸氧化酶的情况一样，发现还原是催化过程中的限速步骤，速率为0.63 s-1。Y129F乙醇酸氧化酶的动力学性质提供了证据，表明酪氨酸129羟基的主要功能是稳定过渡态。
