The P1 reactive site methionine residue of ecotin is not crucial for its specificity on target proteases. A potent inhibitor of pancreatic serine proteases from Escherichia coli.

I.S Seong,H.R Lee,J.H Seol,S.K Park,C.S Lee,S.W Suh,Y.M Hong,M.S Kang,D.B Ha,C.H Chung

doi:10.1016/s0021-9258(17)31889-6

Abstract

The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.

Highlights

The importance of the P,reactive site for the speci- Phe, Leu, and Met inhibit chymotrypsin,and those withPIAla ficity of ecotin on target proteases was examined by and Ser inhibit elastase
Similar ecotin on target proteases, mutagenesis was directed at the results were obtained for chymotrypsin, excethptat its Mets4 residueW. e replaced Cysa' witSher to determine the replacement with Glu caused about 40% reduction of role of the disulfide bond in the ecotin molecule
Less, elastase could be completelyinhibited upon ineu- Single-stranded, uracil-containing phagemids were prepared by infectbation with excess amounts of the mutant ecotin con- ing E. coli strain CJ236 with helper phage R408 [8].Mutataining Arg, Glu,or Tyr

Summary

FXPERIMENTAL PROCEDURES

Dramaticallyreduced its ability to inhibit elastase, Site-directed Mutagenesis-The recombinant Bluescript plasmid while that with Ile showed little or no effect. Inhibition of Target Proteases by Mutant Ecotins-To deter- Since replacementsof Met84even with Glu and Tyr were withmine whether the mutations at the PI reactive site alter the out anyeffect on the inhibitory activity of ecotin against trypspecificity of ecotin or affect its ability to inhibit target pro- sin, we wondered whether or not the mutanftorms of ecotin are teases, theinhibitory activityof the mutanftorms of ecotin was cleaved at the mutatedPI site under the samiencubation concompared to that of the wild-type inhibitor against trypsin, dition. Similar data were obtained for their ability to not exactly match with the size of intact ecotin This result inhibit chymotrypsin, except that the replacement with Glu suggest that an additional cleavage may occur in the ecotin

Mutations of the PI Met in Ecotin

Relative activity of ecotin after heatingfor

Findings

DISCUSSION