Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus.
The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.
Arnone
M I
MI
Dipartimento di Chimica Organica e Biologica, Università di Napoli, Italy.
Birolo
L
L
Giamberini
M
M
Cubellis
M V
MV
Nitti
G
G
Sannia
G
G
Marino
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D016944:Sulfolobus / Q000201:enzymology*; D013552:Swine; D013696:Temperature; D013820:Thermolysin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D001219:Aspartate Aminotransferases; D010447:Peptide Hydrolases; D013820:Thermolysin