乙醇酸氧化酶必需精氨酸残基的化学修饰

Chemical Modification of an Essential Arginyl Residue in Glycolate Oxidase

丁二酮能使GAO迅速失活,其失活速度受介质pH和硼酸浓度的显著影响;其修饰反应具可逆性,当透析除去修饰剂和硼酸时,活性得到恢复。失活进程表现为假一级动力学。而计算表明,酶的每一活性中心单位与一分子丁二酮结合便可引起酶的失活。底物和竞争性抑制剂均能有效地保护酶免于失活。氨基酸分析表明,酶的失活是因为丁二酮修饰了精氨酸残基。丁二酮修饰GAO后使酶的K_m增大,而V_m没有变化。

Treatment of glycolate oxidase (GAO) from Brassica chinensis var. tsaitai with butanedione and glyoxal could markedly decrease its activity. This inactivation depended on the presence of borate (100 mmol/L) and the pH at which the modification reaction proceeded (Fig. 1, 4 and Table 1) The inactivation rate followed the pseudo first order kinetics with respect to reaction time and modifier concentration (Fig. 3A, B and Fig.4). It was demonstrated by the method of Keech and Farrant (1968) that only one molecule of butanedione reacted with each active unit to produce an inactivated enzyme-modifier complex (Fig. 3C). The inactivation was reversed when butanedione and borate were removed by dialysis (Fig. 2). All substrates (glycolate, glyoxylate and lactate) and competitive inhibitors (oxalate and oxaloacetate) could protect GAO against its inactivation by butanedione (Table 2). Amino acid analysis showed that the inactivation of the enzyme was due to the modification of the arginyl residue by butanedione (Table 3). It was found that after the modification of GAO by butanedione K_m (glycolate) value increased while V_(max) remained unchanged (Fig. 5). It is concluded that an arginyl residue is essential for GAO activity, probably acting as a combining site for the substrate.