中国生物化学与分子生物学报
中國生物化學與分子生物學報
중국생물화학여분자생물학보
CHINESE JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
2006年
4期
301-307
,共7页
EPSP合酶%莽草酸途径%稳态动力学%核盘菌%草甘膦
EPSP閤酶%莽草痠途徑%穩態動力學%覈盤菌%草甘膦
EPSP합매%망초산도경%은태동역학%핵반균%초감련
EPSP synthase%shikimate pathway%steady-state kinetics%Sclerotinia sclerotiorum%glyphosate
核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶(EPSP合酶)是AROM多功能酶的活性之一.该酶催化莽草酸磷酸(S3P)和磷酸烯醇式丙酮酸(PEP)产生5-烯醇丙酮酰莽草酸-3-磷酸和无机磷酸的可逆反应,受除草剂草甘膦(N-(膦羧甲基)甘氨酸)抑制.纯化了核盘菌AROM蛋白并对EPSP合酶进行了酶学特征研究.结果显示,该酶反应的最适pH值为7.2,最适温度为30℃.热失活反应活化能是69.62 kJ/mol.底物S3P和PEP浓度分别高于1 mol/L和2 mmol/L时,对EPSP合酶反应产生抑制作用.用双底物反应恒态动力学Dalziel方程求得的Km(PEP)为140.98μmol/L,Km(S3P)为139.58μmol/L.酶动力学模型遵循顺序反应机制.草甘膦是该酶反应底物PEP的竞争性抑制剂(Ki为0.32μmol/L)和S3P的非竞争性抑制剂.正向反应受K+激活.当[K+]增加时,Km(PEP)随之降低,Km(S3P)不规律变化,而Ki(PEP)随[K+]增加而提高.
覈盤菌5-烯醇丙酮酰莽草痠-3-燐痠閤酶(EPSP閤酶)是AROM多功能酶的活性之一.該酶催化莽草痠燐痠(S3P)和燐痠烯醇式丙酮痠(PEP)產生5-烯醇丙酮酰莽草痠-3-燐痠和無機燐痠的可逆反應,受除草劑草甘膦(N-(膦羧甲基)甘氨痠)抑製.純化瞭覈盤菌AROM蛋白併對EPSP閤酶進行瞭酶學特徵研究.結果顯示,該酶反應的最適pH值為7.2,最適溫度為30℃.熱失活反應活化能是69.62 kJ/mol.底物S3P和PEP濃度分彆高于1 mol/L和2 mmol/L時,對EPSP閤酶反應產生抑製作用.用雙底物反應恆態動力學Dalziel方程求得的Km(PEP)為140.98μmol/L,Km(S3P)為139.58μmol/L.酶動力學模型遵循順序反應機製.草甘膦是該酶反應底物PEP的競爭性抑製劑(Ki為0.32μmol/L)和S3P的非競爭性抑製劑.正嚮反應受K+激活.噹[K+]增加時,Km(PEP)隨之降低,Km(S3P)不規律變化,而Ki(PEP)隨[K+]增加而提高.
핵반균5-희순병동선망초산-3-린산합매(EPSP합매)시AROM다공능매적활성지일.해매최화망초산린산(S3P)화린산희순식병동산(PEP)산생5-희순병동선망초산-3-린산화무궤린산적가역반응,수제초제초감련(N-(련최갑기)감안산)억제.순화료핵반균AROM단백병대EPSP합매진행료매학특정연구.결과현시,해매반응적최괄pH치위7.2,최괄온도위30℃.열실활반응활화능시69.62 kJ/mol.저물S3P화PEP농도분별고우1 mol/L화2 mmol/L시,대EPSP합매반응산생억제작용.용쌍저물반응항태동역학Dalziel방정구득적Km(PEP)위140.98μmol/L,Km(S3P)위139.58μmol/L.매동역학모형준순순서반응궤제.초감련시해매반응저물PEP적경쟁성억제제(Ki위0.32μmol/L)화S3P적비경쟁성억제제.정향반응수K+격활.당[K+]증가시,Km(PEP)수지강저,Km(S3P)불규률변화,이Ki(PEP)수[K+]증가이제고.
The 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity of Sclerotinia sclerotiorum is one of the multifunctional enzyme AROM activities, which catalyzes a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate, and is inhibited by the herbicide glyphosate (N-phosphonomethyl glycine). AROM protein has been purified from Sclerotinia sclerotiorum and the EPSP synthase has been analyzed. The results indicated that the optimal pH and temperature of EPSP synthase were 7.2 and 30℃ respectively. The activation energy of the heat-deactivated reaction of the enzyme was found to be 69.62 kJ/mol. Both of the substrates, S3P and PEP, were showed to inhibit the reaction rate when their concentrations exceeded 1 mmol/L and 2 mmol/L respectively. The Km of 140.98 μmol/L for PEP and 139.58 μmol/L for S3P were obtained by Dalziel equation which was a steadystate kinetic equation of the enzymatic reaction with the double substrates. The kinetic pattern of the enzyme was consistent with a sequential mechanism. Inhibition of the EPSP synthase reaction by glyphosate was competitive with respect to PEP, with the Ki 0. 32 μmol/L, and noncompetitive with regard to S3P. Activation by [ K+ ] was observed in the forward reaction. The Km (PEP) was lowered by increasing [ K+ ], while the Km (S3P) changed irregularly and the Ki (PEP) was enhanced.
