Wedekind J E, Frey P A, Rayment I
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705, USA.
Biochemistry. 1995 Sep 5;34(35):11049-61. doi: 10.1021/bi00035a010.
Galactose-1-phosphate uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDP-galactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multiple isomorphous replacement and electron density modification techniques and has been refined to 1.8 A resolution. Enzyme subunits consist of a single domain with the topology of a "half-barrel". The barrel staves are formed by nine strands of antiparallel beta-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the half-barrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, S gamma of a cysteine is modified by beta-mercaptoethanol, which prevents the alpha-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Leu54, Ohe75, Asn77, Asp78, Phe79, and Val108. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations.
1-磷酸半乳糖尿苷酰转移酶催化尿苷二磷酸葡萄糖的5'-单磷酸尿苷部分可逆地转移至1-磷酸半乳糖的磷酸基团上，形成尿苷二磷酸半乳糖。该酶参与半乳糖代谢的勒洛伊尔途径，其缺失是潜在致命疾病半乳糖血症的主要病因。本文报道了来自大肠杆菌的二聚体酶与尿苷二磷酸复合后的三维结构。该结构通过多重同晶置换和电子密度修正技术解析得到，已精修至1.8埃分辨率。酶亚基由一个具有“半桶状”拓扑结构的单一结构域组成。桶壁由九条反平行β-折叠链形成。桶轴大致平行于与每个亚基相关的局部二元轴。两条两亲性螺旋填充半桶，隔离其疏水内部。一个铁原子位于桶的外部，位于亚基界面中心。亚基内与铁的配位类似于由两个组氨酸的赤道配位、一个双齿羧酸盐基团和一个单一轴向组氨酸形成的扭曲四方锥。这种配位稳定了亚基界面，其进一步特征是形成了两个不同于半桶链的分子间“小折叠片”。连接小折叠片链的环有助于活性位点的形成，活性位点位于桶边缘的外表面。桶链的环通过一个结构锌原子连接在一起，该锌原子以对催化至关重要的方式定向局部折叠。在后者的一个环中，半胱氨酸的Sγ被β-巯基乙醇修饰，这阻止了核苷酸的α-磷接近亲核试剂组氨酸166。这种构象似乎并未扰乱通过亮氨酸54、苏氨酸75、天冬酰胺77、天冬氨酸78、苯丙氨酸79和缬氨酸108的侧链介导的与尿嘧啶和核糖部分的相互作用。后几个残基中的几个与人类半乳糖血症有关。目前的结构解释了其中许多突变的有害影响。
