title: The role of the Cys191-Cys220 disulfide bond in trypsin: new targets for engineering substrate specificity.
abstract: The S1 binding site of trypsin is cross-linked by the conserved Cys191-Cys220 disulfide bond. The substitution of Cys191 and Cys220 with Ala decreases the activity of trypsin by 20-200-fold as measured by kcat/K(m) for the hydrolysis of amide substrates; in contrast, ester hydrolysis is decreased by < 10-fold. Similar decreases are observed in the hydrolysis of oligopeptide and single amino acid substrates. This decrease in activity results from a decrease in the acylation rate. The substrate binding and deacylation rate are not affected by the loss of the disulfide bond. C191A/C220A binds BPTI with the same affinity as trypsin, although the affinity of benzamidine is decreased 10-fold and the affinity of leupeptin is decreased 1000-fold. The CD spectrum of C191A/C220A displays significant differences from that of trypsin; these differences most likely result from the loss of the disulfide chromophore, although perturbation of enzyme structure cannot be discounted. The loss of the Cys191-Cys220 disulfide has no effect on the stability of trypsin as measured by urea denaturation. Single and double substitutions of Ser at positions 191 and 220 have a similar activity to C191A/C220A. These results indicate that the Cys191-Cys220 disulfide bond is not essential for the function, structure or stability of trypsin.
authors.affiliation: Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254, USA.
authors.forename: E C
authors.initials: EC
authors.lastname: Wang
authors.forename: S H
authors.initials: SH
authors.lastname: Hung
authors.forename: M
authors.initials: M
authors.lastname: Cahoon
authors.forename: L
authors.initials: L
authors.lastname: Hedstrom
mesh_terms: D000215:Acylation; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D014357:Trypsin; D003545:Cysteine