ROLE OF TYROSINE-129 IN THE ACTIVE-SITE OF SPINACH GLYCOLATE OXIDASE

The enzymatic properties and the three-dimensional structure of spinac h 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 da ta. 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 muta nt does not lower the pK(a) of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volok ita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pK(a) is a bove pH 9. In contrast to wild-type glycolate oxidase, oxalate does no t 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), s uch as stabilization of the anionic flavin semiquinone and formation o f tight N(5)-sulfite adducts, are all conserved in the Y129F mutant pr otein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type a ctivity. The lower turnover number for the mutant of 0.74 s-1 versus 2 0 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 K(m) values of 1.5 mM and 7 muM for glycolate and oxygen, respectively . The K(m) for glycolate is slightly higher than that found for wild-t ype glycolate oxidase (1 mM) whereas the K(m) 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. T he kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilizatio n of the transition state.