A thermally sensitive loop in clostridial glutamate dehydrogenase detected by limited proteolysis.
The structural flexibility and thermostability of glutamate dehydrogenase (GDH) from Clostridium symbiosum were examined by limited proteolysis using three proteinases with different specificities, trypsin, chymotrypsin, and endoproteinase Glu-C. Clostridial GDH resisted proteolysis by any of these enzymes at 25 degrees C. Above 30 degrees C, however, GDH became cleavable by chymotrypsin, apparently at a single site. SDS-PAGE indicated the formation of one large fragment with a molecular mass of approximately 44 kDa and one small one of <10 kDa. Proteolysis was accompanied by the loss of enzyme activity, which outran peptide cleavage, suggesting a cooperative conformational change. Proteolysis was prevented by either of the substrates 2-oxoglutarate or l-glutamate but not by the coenzymes NAD(+) or NADH. Circular dichroism spectroscopy indicated that the protective effects of these ligands resulted from fixation of flexible regions of the native structure of the enzyme. Size-exclusion chromatography and SDS-PAGE studies of chymotrypsin-treated GDH showed that the enzyme retained its hexameric structure and all of its proteolytic fragments. However, circular dichroism spectroscopy and analytical ultracentrifugation showed global conformational changes affecting the overall compactness of the protein structure. Chymotrypsin-catalyzed cleavage also diminished the thermostability of GDH and the cooperativity of the transition between its native and denatured states. N-terminal amino acid sequencing and mass spectrometry showed that heat-induced sensitivity to chymotrypsin emerged in the loop formed by residues 390-393 that lies between helices alpha(15) and alpha(16) in the folded structure of the enzyme.
Aghajanian
Suren
S
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
Hovsepyan
Manushak
M
Geoghegan
Kieran F
KF
Chrunyk
Boris A
BA
Engel
Paul C
PC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002918:Chymotrypsin / Q000494:pharmacology; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017510:Protein Folding; D020836:Protein Structure, Quaternary
D001426:Bacterial Proteins; D005969:Glutamate Dehydrogenase; D002918:Chymotrypsin