Wilk Sherwin, Wilk Elizabeth, Magnusson Ronald P
Department of Pharmacology, Box 1215, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA.
Arch Biochem Biophys. 2002 Nov 15;407(2):176-83. doi: 10.1016/s0003-9861(02)00494-0.
Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.
天冬氨酰氨肽酶（DAP）是一种广泛分布且含量丰富的胞质酶，可从含N端酸性氨基酸的肽中去除谷氨酰基或天冬氨酰基残基。DAP是共催化金属肽酶MH家族M18家族的成员。人类和小鼠的该酶已被克隆。我们鉴定出8个高度同源的真核序列，它们可能是天冬氨酰氨肽酶。人类DAP的8个组氨酸残基依次突变为苯丙氨酸。His94、His170和His440的突变消除了酶活性。通过与MH家族其他成员的同源性推测，His94和His440参与结合共催化锌原子。His352的突变显著降低了酶活性。对His352突变体的凝胶过滤分析显示四级结构不稳定，天然的440 kDa酶解离。His33以及位于349、359和363位残基簇中的组氨酸的突变均降低了催化常数（k(cat)）。这些研究揭示了组氨酸残基在DAP的催化作用和结构完整性方面的重要作用。
