Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.丙酮酸激酶（pyruvate kinase）通过作为糖酵解的核心调控因子，在葡萄糖细胞代谢中发挥关键作用。该四聚体酶可被多种效应分子（主要为磷酸糖）实施别构调控。当效应分子与底物结合后，已有研究在多种丙酮酸激酶的结构中观测到显著的构象变化。隐孢子虫（Cryptosporidium parvum）的丙酮酸激酶在已知原生动物来源的酶中独具特殊性：在常见效应分子存在的条件下，其不表现出任何别构调控特性。隐孢子虫丙酮酸激酶的晶体结构已通过分子置换法完成解析，并精修至2.5 Å分辨率。其活性位点内，甘油分子定位于三磷酸腺苷（ATP）的γ磷酸位点附近，且该蛋白结构呈现出部分闭合的活性位点形态。然而，与其他活性位点呈闭合状态的丙酮酸激酶结构不同，隐孢子虫丙酮酸激酶中的α6'螺旋发生解旋，呈现出伸展构象。在该晶体结构中，一个硫酸根离子结合于一个保守位点——该位点在多数丙酮酸激酶结构中为效应分子的磷酸基团所占据。隐孢子虫丙酮酸激酶结构的一项新特征是：在不对称单元内的两个单体之间存在二硫键交联。该二硫键由一个单体的短N端螺旋上的26号半胱氨酸残基，与另一个单体的长螺旋（残基303–320）上的312号半胱氨酸残基在单体界面处形成。这两个半胱氨酸残基均为隐孢子虫所特有，且该二硫键在还原环境中仍可保持完整。不过截至目前，该二硫键的生物学意义（若确实存在的话）仍未明确。