The structural basis of proteolytic activation of bovine glutamate dehydrogenase.
In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that 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 nontreated chymotrypsin may have led to the production of the larger 4-5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.
Carrigan
John B
JB
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
Engel
Paul C
PC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D015842:Serine Proteinase Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D012710:Serum Albumin, Bovine; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase; D002918:Chymotrypsin; D014357:Trypsin