乙醇酸氧化酶的纯化结晶和酪氨酸残基的修饰对酶活性的影响

Purification and Crystallization of Glycolate Oxidase and Effect of Modification of Tyrosine Residue on Its Activity

从青菜叶片中纯化的GAO为淡黄色棒状或针状结晶,纯化度达129.3倍。NBS对GAO有强烈的抑制作用,其抑制效应不受pH的影响,抑制程度和 NBS浓度(0～10 μmol/L)呈线性关系;乙醇酸和草酸均能保护GAO免受NBS的抑制。NBS在全抑制浓度范围内并不降低OD_(280),但引起OD_(260)明显上升;当用乙醇酸保护GAO时,这种上升与酶的抑制同时消失。另外两种更专一的酪氨酸残基修饰剂NBSF和N—AI亦显著抑制GAO活性,底物乙醇酸对此有保护作用,N—AI抑制的GAO可被羟胺复活。

Glycolate oxidase (GAO) was purified andcrystallized from leaves of Brassica chinensis var.tsai-tai to a purification factor of 129.3. Thecrystals were yellowish in colour and were of theshape of rod and needle. GAO was markedly inhibited by 10 umol/LNBS in Tris-HC1 buffer (pH 8.3). The inhi-bition was directly proportional to NBS con-centrations in the range of 0～10 umol/L (mo-dification for 10 min). Changes in pH values ofthe medium did not significantly affect the in-hibition. Glycolate and oxalate could protectthe enzyme against the inhibition by NBS.Spectrophotometric observations showed thatthe absorption peak at 280 nm of modified en-zyme was not lowered as would happen whentryptophan was oxidited, while the absorptionat 260 nm was markedly increased by the modi-fication of enzyme with NBS. This indicated thatthe okidation of tyrosine residue was involvedin modification. The increase of absorption at260 nm was accompanied by a simultaneous andparallel inhibition of GAO. Both the inhibitionand the increase of absorption at 260 nm werediminished when the enzyme was protected byits substrate glycolate. NBSF and N-AI, twospecific modifiers of tyrosine residue, markedlyinhibited the activity of GAO. Glycolate couldprotect GAO against the inhibition. The enzymeinhibited by N-AI could be reactivated by 0.3mol/L hydroxylamine (30 min). From theseresults we conclude that the inhibition of GAOby NBS, NBSF and N-AI is due to the modi-fication of the tyrosine residue in the active cen-ter of the enzyme, and that tyrosine is essentialto the activity of GAO.