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THE STRUCTURAL BASIS OF PROTEOLYTIC ACTIVATION OF BOVINE GLUTAMATE DEHYDROGENASE

University College Dublin

(RECEIVED February 4, 2008; ACCEPTED April 24, 2008)

In this work, we re-examine the previously-reported phenomenon of the creation of a super-active glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies which came to light during those studies. We find that superactivation is caused by cleavage at the N-terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in non-treated chymotrypsin may have led to the production of the larger 4-5kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous papers, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis while ADP activation is almost abolished. Utilising the recently-solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.

Keywords: Enzymes; Structure/function studies; Peptide/fragment isolation; Kinetics; enzyme activation; partial proteolysis

¹ E-mail: paul.engel{at}ucd.ie

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