Purification, characterization and cDNA sequence of an alkaline chymotrypsin from the midgut of Manduca sexta.
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.
Peterson
A M
AM
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Fernando
G J
GJ
Wells
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004064:Digestive System / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D002918:Chymotrypsin