PMID: 9194165Apr 1, 1997Paper

The role of the Cys191-Cys220 disulfide bond in trypsin: new targets for engineering substrate specificity

Protein Engineering
E C WangL Hedstrom

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 d...Continue Reading

Citations

Mar 19, 1999·Protein Science : a Publication of the Protein Society·A Vindigni, E Di Cera
Jul 24, 1998·Biochimica Et Biophysica Acta·J PolanowskaJ Otlewski
Apr 2, 1999·European Journal of Biochemistry·H Czapinska, J Otlewski
Apr 24, 2008·The FEBS Journal·Shunsuke KawamuraTakao Torikata
May 5, 2006·Protein Science : a Publication of the Protein Society·Teaster T BairdCharles S Craik
Aug 26, 2009·Cell·Najeeb HalabiRama Ranganathan
Jul 20, 2007·The Journal of Biological Chemistry·Leslie A Bush-PelcEnrico Di Cera
Oct 22, 2013·The Journal of Biological Chemistry·Kristina M CookPhilip J Hogg
Sep 19, 2003·Biochemical and Biophysical Research Communications·Erzsébet KénesiLászló Szilágyi
Jan 3, 2020·Journal of Chemical Information and Modeling·Marcelo Querino Lima AfonsoLucas Bleicher

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