Evidence for a catalytic dyad in the active site of homocitrate synthase from Saccharomyces cerevisiae.
Evidence for a catalytic dyad in the active site of homocitrate synthase from Saccharomyces cerevisiae.
复制标题
酿酒酵母高柠檬酸合酶活性位点催化二元体的证据。
DOI：
10.1021/bi800087k
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Cook,PaulF
中科院分区：
文献类型：
--
作者：
Qian,Jinghua;Khandogin,Jana;West,AnnH;Cook,PaulF
高柠檬酸合成酶(乙酰辅酶A：2-酮戊二酸C-转移酶；E.C.2.3.3.14)催化乙酰辅酶A(AcCoA)和α-酮戊二酸(α-KG)缩合生成高柠檬酸和辅酶A。虽然HCS的结构还没有解决，但苹果酸异丙酯合成酶(IPMS)的结构已经解决(Koon，N.，Squire，C.J.和Baker，E.N.(2004)Proc.娜塔莉。阿卡德。SCI。美国101,8295−8300)。三个活性中心残基Glu-218、His-379和Tyr-410被认为是参与AcCoA甲基去质子化的催化残基，在Claisen缩合生成高辛基CoA之前。IPMS中的三个活性中心残基在酿酒酵母的HCS中都是保守的。用定点突变的方法研究了同源残基Glu-155、His-309和Tyr-320在THs中的作用。酿酒人。未检测到H309A和H309N突变酶的活性，但在300 mM咪唑存在下，H309A的活性略有增加，但仍比野生型(Wt)低1000倍。E155Q和E155A突变酶的活性比wt低1000倍，E155A的活性可被甲酸盐部分挽救，但E155Q的活性不能被甲酸盐部分挽救，其最大活性约为60 mM，最大活性仅为wt的4倍。在甲酸盐存在下，E155A的KCAT、KAcCoA和Kα-KG值分别为0.0031 S−1、13μM和39μM。在甲酸盐存在下，E155A的kcat的pH依赖性给出了一个基团的pKaof为7.9，该基团必须被质子化才能获得最佳活性，与wt酶的观察到的结果相似。然而，在剖面的酸性侧观察到了部分变化，而对于wt观察到的全部或无变化给出的pKa值约为6.7。E155Q的kcat值在高pH时下降，与wt酶相似，但在低pH时不依赖于pH。Y320F突变体酶活仅为wt的25倍，其中KCAT、KAcCoA和Kα-KG值分别为0.039 S−1、33μM和140μM，对V/KAcCoA和V的一级动力学氘同位素效应分别为1.30和1.8.这些数据与wt相似。这些数据结合恒定pH的分子动力学模拟研究表明，由Glu-155和His-309组成的催化二聚体起去AcCoA甲基的作用，而Tyr320可能不直接参与催化，但可能有助于定向反应物和/或催化动力学。
Homocitrate synthase (acetyl-coenzyme A: 2-ketoglutarate C-transferase; E.C. 2.3.3.14) (HCS) catalyzes the condensation of acetyl-CoA (AcCoA) and α-ketoglutarate (α-KG) to give homocitrate and CoA. Although the structure of an HCS has not been solved, the structure of isopropylmalate synthase (IPMS), a homologue, has been solved (Koon, N., Squire, C. J., and Baker, E. N. (2004)Proc. Natl. Acad. Sci. U.S.A. 101, 8295−8300). Three active site residues in IPMS, Glu-218, His-379, and Tyr-410, were proposed as candidates for catalytic residues involved in deprotonation of the methyl group of AcCoA prior to the Claisen condensation to give homocitrylCoA. All three of the active site residues in IPMS are conserved in the HCS fromSaccharomyces cerevisiae. Site-directed mutagenesis has been carried out to probe the role of the homologous residues, Glu-155, His-309, and Tyr-320, in theS. cerevisiaeHCS. No detectable activity was observed for the H309A and H309N mutant enzyme, but a slight increase in activity was observed for H309A in the presence of 300 mM imidazole, which is still 1000-fold lower than that of wild type (wt). The E155Q and E155A mutant enzymes exhibited 1000-fold lower activity than wt. The activity of E155A, but not of E155Q, could be partially rescued by formate; aKactof 60 mM with a modest 4-fold maximum activation was observed. In the presence of formate, E155A giveskcat,KAcCoA, andKα-KGvalues of 0.0031 s−1, 13 μM, and 39 μM, respectively, while a primary kinetic deuterium isotope effect of about 1.4 was obtained onV, with deuterium in the methyl of AcCoA. The pH dependence ofkcatfor E155A in the presence of formate gave a pKaof 7.9 for a group that must be protonated for optimum activity, similar to that observed for the wt enzyme. However, a partial change was observed on the acid side of the profile, compared to the all or none change observed for wt giving a pKaof about 6.7. Thekcatfor E155Q decreased at high pH, similar to the wt enzyme, but was pH independent at low pH. The Y320F mutant enzyme only lost 25-fold activity compared to that of the wt, givingkcat,KAcCoA, andKα-KGvalues of 0.039 s−1, 33 μM, and 140 μM, respectively, and a primary kinetic deuterium isotope effect of 1.3 and 1.8 onV/KAcCoAandV, respectively; the pH dependence ofkcatwas similar to that of the wt. These data, combined with a constant pH molecular dynamics simulation study, suggest that a catalytic dyad comprising Glu-155 and His-309 acts to deprotonate the methyl group of AcCoA, while Tyr320 is likely not directly involved in catalysis, but may aid in orienting the reactant and/or the catalytic dyad.