Duffel M W, Chen G, Sharma V
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City 52242, USA.
Chem Biol Interact. 1998 Feb 20;109(1-3):81-92. doi: 10.1016/s0009-2797(97)00122-1.
Active site-directed affinity labeling was utilized to elucidate peptide sequences at the binding site for sulfuryl acceptors in rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9). The affinity labeling reagent, N-bromoacetyl-4-hydroxyphenylamine, was designed on the basis of substrate specificity studies with para-substituted phenols, utilization of a bromoacetamido group for reactivity with active site amino acid residues and its similarity to acetaminophen, a known substrate for aryl (phenol) sulfotransferases. AST IV utilized N-bromoacetyl-4-hydroxyphenylamine as a substrate with kinetic constants that compared favorably to those obtained with acetaminophen. Incubation of AST IV with N-bromoacetyl-4-hydroxyphenylamine at pH 7.0 in the absence of PAPS and other substrates resulted in an irreversible inactivation of the enzyme that was both time- and concentration-dependent. [14C]-N-bromoacetyl-4-hydroxyphenylamine was synthesized and used to analyze the regions of protein sequence that were involved in the binding of the affinity label. AST IV was incubated with [14C]-N-bromoacetyl-4-hydroxyphenylamine, hydrolyzed with endoproteinase Lys-C and the labeled peptides were purified by HPLC. Control incubations of AST IV with the affinity label in the presence of 4-propylphenol and PAP were utilized to ascertain the specificity of the interaction. Sequence analysis of the labeled peptides, carried out by automated Edman degradation, revealed labeling sites on cysteine (Cys-232, Cys-283 and Cys-289) and lysine (Lys-286) residues near the C-terminus of the protein. The locations of these labeling sites were further evaluated both by sequence-alignment with other sulfotransferases and by theoretical calculations on predicted secondary structure.
利用活性位点导向的亲和标记法来阐明大鼠肝脏芳基硫酸转移酶（AST）IV（也称为酪氨酸酯硫酸转移酶，EC 2.8.2.9）中硫酸基受体结合位点的肽序列。亲和标记试剂N-溴乙酰基-4-羟基苯胺是根据对对位取代酚的底物特异性研究、利用溴乙酰胺基与活性位点氨基酸残基反应以及它与对乙酰氨基酚（一种已知的芳基（酚）硫酸转移酶底物）的相似性设计的。AST IV将N-溴乙酰基-4-羟基苯胺用作底物，其动力学常数与用对乙酰氨基酚获得的常数相当。在不存在3'-磷酸腺苷-5'-磷酰硫酸（PAPS）和其他底物的情况下，将AST IV与N-溴乙酰基-4-羟基苯胺在pH 7.0下孵育，导致酶的不可逆失活，这是时间和浓度依赖性的。合成了[14C]-N-溴乙酰基-4-羟基苯胺并用于分析参与亲和标记结合的蛋白质序列区域。将AST IV与[14C]-N-溴乙酰基-4-羟基苯胺孵育，用内肽酶Lys-C水解，然后通过高效液相色谱法纯化标记的肽。在4-丙基酚和PAP存在下，将AST IV与亲和标记进行对照孵育，以确定相互作用的特异性。通过自动Edman降解对标记肽进行序列分析，揭示了蛋白质C末端附近的半胱氨酸（Cys-232、Cys-283和Cys-289）和赖氨酸（Lys-286）残基上的标记位点。通过与其他硫酸转移酶进行序列比对以及对预测二级结构进行理论计算，进一步评估了这些标记位点的位置。
