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, cha racterized and the cDNA encoding the protein has been cloned. The enzy me exists as a monomer of approx, 24 kDa and shows maximal activity be tween pH 10.5 and 11.0, Kinetic studies reveal that the Michaelis cons tant (K-m) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-ni troanilide varies only slightly between pH 7.5 and 11.5 and the Dixon plot shows a kinetically significant pK(a) at 9.2, The specificity of the purified enzyme was determined to be the peptide bond on the carbo xyl side of tyrosine, phenylalanine, tryptophan, histidine, leucine, t hreonine and glycine. The protease is inhibited by TPCK, PMSF, chymost atin and DFP. A 1 kilobase chymotrypsin cDNA clone was isolated and se quenced. The cDNA sequence encodes a preproenzyme with a putative 17 a mino acid signal sequence, a 41 amino acid activation peptide and a ma ture 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 local izes the mRNA for the chymotrypsin to the anterior and middle third of the midgut.