title: Converting trypsin to elastase: substitution of the S1 site and adjacent loops reconstitutes esterase specificity but not amidase activity.
abstract: The conversion of trypsin into a protease with chymotrypsin-like activity and specificity required substitution of fifteen residues in the S1 site and two surface loops with their chymotrypsin counterparts [Hedstrom,L., Szilagyi,L. and Rutter,W.J. (1992) Science, 255, 1249-1253]. These residues may define a set of general structural determinants of specificity in the trypsin family. In order to test this hypothesis, we have attempted to convert trypsin into a protease with specificity for substrates containing small aliphatic residues by replacing the S1 site and these surface loops with the analogous residues of elastase. Five elastase-like mutant enzymes were constructed with various combinations of these substitutions. Four mutant enzymes catalyze the hydrolysis of MeOSuc-Ala-Ala-Pro-Ala-SBzl more efficiently than the hydrolysis of Suc-Ala-Ala-Pro-Phe-SBzl. This observation indicates that the mutant enzymes have elastase-like esterase specificity. The best mutant, Tr-->E1-2, is a more specific esterase than elastase: the ratio of the values of kcat/Km for MeOSuc-Ala-Ala-Pro-Ala-SBzl and Suc-Ala-Ala-Pro-Phe-SBzl is greater than 160 for Tr-->E1-2 and 50 for elastase. However, the esterase activity of Tr-->E1-2 is 300-fold less than elastase; in addition, Tr-->E1-2 has no measurable amidase activity. Thus these substitutions do not construct a protease with elastase-like activity. These experiments indicate that a unique structural solution is required for each different specificity. Previous work suggested that instability of the S1 site is a major barrier to redesigning the specificity of trypsin. This view is corroborated by preliminary structural studies of Tr-->E1-2. One dimensional 1H NMR spectrum of Tr-->E1-2 suggests that the S1 site and the two surface loops of this mutant trypsin may be disordered.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02254-9110, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Hung
authors.forename: L
authors.initials: L
authors.lastname: Hedstrom
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D010196:Pancreatic Elastase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D004950:Esterases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D000581:Amidohydrolases; C019665:amidase