Study on the Alteration of Catalytic Activities by the Change of Three Dimensional Structure of Enzyme Proteins

查尔酮合成酶（Chalcone synthase，CHS）是植物源聚酮化合物合成酶的代表，催化类黄酮生物合成的第一步反应形成其基本骨架。CHS广泛存在于植物中，在保护植物免受外界胁迫方面发挥着重要的生理作用。与查耳酮合酶相关的其他聚酮脱氢酶具有很高的同源性，称为查耳酮合酶超家族。CHS的反应与脂肪酸合成酶（FAS）非常相似，但其独特的酶反应是丙二酰辅酶A与FAS中的ACP丙二酰辅酶A的脱羧直接缩合。小分子量的CHS（约. 43KD）是最有吸引力的研究点。1.利用E.利用大肠杆菌、酵母和空泡病毒等多种表达系统大量表达Chess，并对不同表达系统表达的CHIS的反应性进行了研究。2.我们首先证明了查耳酮和二苯乙烯脱氢酶之间存在交叉反应，即两种酶产生的副产物是对应的酶产物。当在真核系统中表达时，交叉反应的程度降低。3.裸盖蕨是产生苯基吡喃酮的最原始的维管植物。获得了CHIS超家族的5个克隆，并对其反应进行了表征。除了CHIS，两个STS和异戊苯酮合酶，它含有无功能的CHIS家族mRNA。聚酮合酶中的必需组氨酸被谷氨酰胺和回复突变体取代，如CHIS和STS中谷氨酰胺突变为组氨酸，未显示聚酮合酶的任何显著反应。酶活性的降低是由于其他氨基酸的改变影响了整个三维结构的变化。

Chalcone synthase (CHS) is the representative polyketide synthase of plant origin, which catalyzes the first reaction of flavonoid biosynthesis to form its basic skeleton. CHSs are ubiquitously present in plants and play essential physiological role to protect plants from outer stresses. The other polyketide synthases relating to chalcone synthase have a high homology and called chalcone synthase superfamily. The reaction CHS is very similar to fatty acid synthase (FAS), however its unique enzyme reaction is the direct condensation of malonyl CoA with decarboxylation in contrast to ACP malonyl CoA in FAS. Small molecular weight of CHS (ca. 43KD) is the most attractive point of study. 1. Heterologous expression sytems with E. coli, yeast and vaculovirus were established to obtain Chess in a large amount and reactions of CHIS obtained by different expression systems were investigated in details. 2. We first demonstrate that chalcone and stilbene synthases showed cross reactions, that is the two enzyme produced counter part enzyme products as by products. The degree of cross reaction was decreased when the enzymes were expressed in eukaryote systems. 3. Psilotum nodum is the most primitive vascular plant which produces phnylpyrone. Five clones of CHIS superfamily were obtained and their reaction was characterized. In addition to CHIS, two STS and isovalerophenone synthase it contains unfunctional CHIS family mRNA. Essential histidine in polyketide synthase is replaced with glutamine and revertant, mutated glutamine to histidine as in CHIS and STS did not show any significant reaction of polyketide synthase. The lack of enzyme activity is due to the change of whole three dimentional structure which is affected by the alteration of other amino acids.