Evans C T, Kurz L C, Remington S J, Srere P A
Department of Veterans Affairs Medical Center, University of Texas Southwestern Medical Center at Dallas, USA.
Biochemistry. 1996 Aug 20;35(33):10661-72. doi: 10.1021/bi960336e.
We examined the catalytic efficiency of 18 pig citrate synthase mutants. The residues mutated were selected according to two criteria: the conservation of that residue in all known citrate synthase sequences, and the importance of that residue in substrate-amino acid interactions suggested by the extensive crystal structure information on the enzyme and its complexes. Several changes were made at certain residues to probe the effects of size, hydrogen bonding, and charge on the kinetics of the enzyme. The mutations, as expected, affected the kcats and Kms for OAA and acetyl-CoA to varying degrees. The catalytic efficiency of each of the mutants was determined by the kcat/Km for the individual substrates, OAA and acetyl-CoA. All mutations affected kcat. There was only one mutant, Asp327 Asn, in which the Kms primarily were affected. Most mutations affected both kcat and Km and included the following: His274Gly, His274Arg, Asp375Gly, Asp375Asn, Asp375Glu, Asp375Gln, His320Gly, His320Gln, His320Asn, His320Arg, Arg401His, Gly275Val, and Gly275Ala. The mutations, Arg401Gly, Arg401Lys, His235Gln, and Asn242Glu, had smaller effects on kcat and Km. The CS mutant Arg401Lys exhibited a modestly improved kcat/Km for both substrates compared to the nonmutant enzyme. X-ray crystallographic studies at 2.7 A resolution of one of the mutants, His274Gly, have been undertaken. The mutant enzyme crystallizes in an "open" conformation essentially isomorphous to wild type. The refined model has good geometry and a crystallographic R factor of 0.187 for 11 441 reflections observed between 6.0 and 2.7 A resolution. The refined model revealed a localized relaxation of the structure to relieve strain imposed by a high-energy main and side chain conformation of His274 in the nonmutant, but otherwise the mutation does not result in major structural alterations. Preliminary electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an "enolized" version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible role for the catalytically essential His274 is to stabilize this by charge delocalization mediated by a hydrogen bond. These results provide the basis for further studies of the effects of these changes on the several reactive intermediates, activated substrates, and transition states which may occur along the reaction coordinate for this type of Claisen enzyme.
我们检测了18种猪柠檬酸合酶突变体的催化效率。根据两个标准选择突变的残基：该残基在所有已知柠檬酸合酶序列中的保守性，以及基于该酶及其复合物的大量晶体结构信息所表明的该残基在底物 - 氨基酸相互作用中的重要性。在某些残基处进行了几处改变，以探究大小、氢键和电荷对酶动力学的影响。正如预期的那样，这些突变不同程度地影响了草酰乙酸（OAA）和乙酰辅酶A的催化常数（kcats）和米氏常数（Kms）。每个突变体的催化效率由单个底物OAA和乙酰辅酶A的催化常数与米氏常数的比值（kcat/Km）来确定。所有突变都影响了催化常数。只有一个突变体，即天冬氨酸327突变为天冬酰胺（Asp327 Asn），其米氏常数受到主要影响。大多数突变同时影响催化常数和米氏常数，包括以下突变体：组氨酸274突变为甘氨酸（His274Gly）、组氨酸274突变为精氨酸（His274Arg）、天冬氨酸375突变为甘氨酸（Asp375Gly）、天冬氨酸375突变为天冬酰胺（Asp375Asn）、天冬氨酸375突变为谷氨酸（Asp375Glu）、天冬氨酸375突变为谷氨酰胺（Asp375Gln）、组氨酸320突变为甘氨酸（His320Gly）、组氨酸320突变为谷氨酰胺（His320Gln）、组氨酸320突变为天冬酰胺（His320Asn）、组氨酸320突变为精氨酸（His320Arg）、精氨酸401突变为组氨酸（Arg401His）、甘氨酸275突变为缬氨酸（Gly275Val）以及甘氨酸275突变为丙氨酸（Gly275Ala）。精氨酸401突变为甘氨酸（Arg401Gly）、精氨酸401突变为赖氨酸（Arg401Lys）、组氨酸235突变为谷氨酰胺（His235Gln）以及天冬酰胺242突变为谷氨酸（Asn242Glu）这些突变对催化常数和米氏常数的影响较小。与未突变的酶相比，柠檬酸合酶突变体精氨酸401突变为赖氨酸（CS mutant Arg401Lys）对两种底物的催化常数与米氏常数的比值有适度提高。已对其中一个突变体组氨酸274突变为甘氨酸（His274Gly）进行了分辨率为2.7埃的X射线晶体学研究。该突变酶以一种“开放”构象结晶，与野生型基本同晶型。对于在6.0至2.7埃分辨率之间观察到的11441个反射，精修模型具有良好的几何结构，晶体学R因子为0.187%。精修模型显示结构发生局部松弛，以缓解未突变体中组氨酸274的高能主链和侧链构象所施加的应变，但除此之外，该突变不会导致主要的结构改变。初步的静电计算支持了这样一种概念，即柠檬酸合酶催化反应限速步骤中的过渡态可能是乙酰辅酶A的一种“烯醇化”形式，它既不是中性的也不是完全带负电荷的，并且催化必需的组氨酸274的一个可能作用是通过氢键介导的电荷离域来稳定这种状态。这些结果为进一步研究这些变化对几种反应中间体、活化底物以及可能出现在这类克莱森酶反应坐标上的过渡态的影响提供了基础。
