Peterson A M, Fernando G J, Wells M A
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Insect Biochem Mol Biol. 1995 Jul;25(7):765-74. doi: 10.1016/0965-1748(94)00092-v.
The chymotrypsin in the midgut of Manduca sexta has been purified, characterized and the cDNA encoding the protein has been cloned. The enzyme exists as a monomer of approx. 24 kDa and shows maximal activity between pH 10.5 and 11.0. Kinetic studies reveal that the Michaelis constant (Km) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide varies only slightly between pH 7.5 and 11.5 and the Dixon plot shows a kinetically significant pKa at 9.2. The specificity of the purified enzyme was determined to be the peptide bond on the carboxyl side of tyrosine, phenylalanine, tryptophan, histidine, leucine, threonine and glycine. The protease is inhibited by TPCK, PMSF, chymostatin and DFP. A 1 kilobase chymotrypsin cDNA clone was isolated and sequenced. The cDNA sequence encodes a preproenzyme with a putative 17 amino acid signal sequence, a 41 amino acid activation peptide and a mature enzyme of 235 amino acids. The isolated clone encodes the highly conserved active site residues (His, Asp, Ser) and specificity pocket residues present in bovine chymotrypsinogen B. Northern analysis localizes the mRNA for the chymotrypsin to the anterior and middle third of the midgut.
烟草天蛾中肠的胰凝乳蛋白酶已被纯化、表征，并且编码该蛋白质的cDNA已被克隆。该酶以约24 kDa的单体形式存在，在pH 10.5至11.0之间表现出最大活性。动力学研究表明，合成底物N-琥珀酰-Ala-Ala-Pro-Phe对硝基苯胺的米氏常数（Km）在pH 7.5至11.5之间变化很小，狄克逊图显示在9.2处有一个动力学上显著的pKa。纯化酶的特异性被确定为酪氨酸、苯丙氨酸、色氨酸、组氨酸、亮氨酸、苏氨酸和甘氨酸羧基侧的肽键。该蛋白酶被TPCK、PMSF、抑肽酶和DFP抑制。分离并测序了一个1千碱基的胰凝乳蛋白酶cDNA克隆。该cDNA序列编码一个前体酶原，带有一个推定的17个氨基酸的信号序列、一个41个氨基酸的激活肽和一个235个氨基酸的成熟酶。分离的克隆编码牛胰凝乳蛋白酶原B中存在的高度保守的活性位点残基（His、Asp、Ser）和特异性口袋残基。Northern分析将胰凝乳蛋白酶的mRNA定位到中肠的前三分之一和中间三分之一处。
