探索在fructose1衬底绑定和歧视,6-bisphosphate和塔格糖1,6-bisphosphate醛缩酶。
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Zgiby SM,汤姆森GJ, Qamar年代,浆果
探索在fructose1衬底绑定和歧视,6-bisphosphate和塔格糖1,6-bisphosphate醛缩酶。
3月。2000欧元;267 (6):1858 - 68。
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10712619 (在PubMed]
- 文摘
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果糖1,6-bisphosphate醛缩酶催化作用可逆glycerone-P凝结和甘油醛3 -磷酸果糖1,6-bisphosphate。最近的大肠杆菌二级结构果糖1,6-bisphosphate醛缩酶(大厅,湄伦纳德,G.A.、苇、C.D.、瓦特、C.I.,浆果,a &猎人,W.N. (1999) j·摩尔。杂志。287年,383 - 394年)在过渡态类似物的存在phosphoglycolohydroxamate划定个别氨基酸的角色绑定glycerone-P和最初的质子抽象步骤的机制。x射线结构已经被使用,加上序列比对,定点诱变和稳态酶动力学扩展这些研究映射重要的残基甘油醛3 -磷酸的绑定。从这些研究三个残留物(Asn35 Ser61和Lys325)在催化已经被确认为重要。我们表明,突变Ser61丙氨酸增加了果糖的Km值1,6-bisphosphate 16倍和产物抑制的研究表明,这种影响是表现最强烈甘油醛3 -磷酸结合口袋的活性部位,证明Ser61参与绑定甘油醛3 -磷酸。相比之下S61T突变没有影响催化强调一个羟基,这一角色的重要性。突变的Asn35 (N35A)导致酶只有1.5%的野生型酶的活动和不同的局部反应表明,该绑定两个丙糖底物的残留的影响。最后,Lys325的突变对催化的影响大于对绑定,但是,考虑到它可能影响的大小具有间接作用在维护其他重要残留在催化地主管构象。有趣的是,尽管它离活性部位和高序列保护,更换第四残渣,Gln59 (Q59A)没有显著影响酶的功能。 In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.