Galadari Sehamuddin, Wu Bill X, Mao Cungui, Roddy Patrick, El Bawab Samer, Hannun Yusuf A
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Biochem J. 2006 Feb 1;393(Pt 3):687-95. doi: 10.1042/BJ20050682.
Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Dypc1Dydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.
中性神经酰胺酶是新发现的在细胞调节中起重要作用的酶，但对其催化机制了解甚少。在本研究中，全长人中性神经酰胺酶被克隆并在缺乏酵母神经酰胺酶YPC1p和YDC1p的酵母双敲除菌株Dypc1Dydc1中表达。人中性神经酰胺酶的生化特性表明，该酶表现出典型的米氏动力学，在pH 7.5时具有最佳活性。Na⁺和Ca²⁺可增强其活性。Mg²⁺和Mn²⁺有一定的刺激作用，但Zn²⁺、Cu²⁺和Fe²⁺会抑制该酶。二硫苏糖醇和2-巯基乙醇对中性神经酰胺酶的抑制作用呈剂量依赖性。为了确定哪些氨基酸参与中性神经酰胺酶的催化作用，对纯化的酶进行了化学修饰。观察到丝氨酸残基修饰剂二异丙基氟磷酸酯对活性的抑制作用呈剂量依赖性，这表明催化作用中有丝氨酸残基参与。通过对不同物种中性神经酰胺酶序列的比对，选择所有保守的丝氨酸残基进行定点诱变。在六个被突变为丙氨酸的比对丝氨酸残基中，只有S354A突变体完全丧失了活性。Ser354位于一个非常保守的六肽序列GDVSPN内，该序列本身处于一个更大的保守序列NXGDVSPNXXGP/XXC的中间。此外，将共有序列中的Asp352和Cys362突变为丙氨酸会导致中性神经酰胺酶活性丧失。因此，本研究确定了一个新的酰胺酶序列，其中包含一个关键的丝氨酸残基，该丝氨酸残基可能在对神经酰胺中存在的酰胺键的水解攻击中作为亲核试剂发挥作用。
