Document Type

Poster

Publication Date

6-2001

Place of conference

Whitefish, Montana

Conference Sponsor

FASEB

Abstract/ Summary

These studies aimed to test the hypothesis that calcium regulates calpain activity by a mechanism analogous to that used by Ca2+- calmodulin (CaM) to regulate its target enzymes. Site directed mutagenesis of a putative IQ motif, IQ413xxxR417 generated catalytic subunits modified at both, or each, of the key residues of the motif to yield ISxxxQ, ISxxxR, and IQxxxQ. Each of the mutant catalytic subunits was co-expressed with a truncated (21k) small subunit in E. coli. The heterodimeric enzymes were purified by standard methods suggesting each was properly folded. Electrophoresis under native conditions, in the absence of calcium, showed no differences between wt and enzyme mutated at R417Q. Each of these enzymes was also stable to incubation at 45 ºC. In contrast enzymes containing Q413S were resolved into multiple bands on native gels and were not stable at 45 ºC. Two calcium dependent activities were assessed: casein hydrolysis and autoproteolysis. Each of the mutants retained significant autolytic activity but lost much of their caseinolytic activity. We propose two interpretations of the results based in part on the calcium-free structures determined for calpain-2 (Hosfield et al , 1999 EMBO J. 18, 6880 and Strobl et al 2000, PNAS 97, 588). The ability of the enzymes to autoproteolyze suggests that the conformational change required for alignment of the catalytic residues is unimpaired in the mutants. Impaired casein hydrolysis may result from 1) inability to release product after cleavage or 2) failure to form a required substrate binding exosite. The latter concept is consistent with many previously known calpain attributes. Exosites provide a mechanism for achieving high substrate specificity amongst enzymes that share conserved active sites, such as those of the thrombin family and perhaps the calpain family. Confirmation and definition of such exosites will be important as they could provide targets for design of isoform selective inhibitors.

Version

pre-print (i.e. pre-refereeing)

Creative Commons License

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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