Hysteretic behaviour of citrate synthase. Site-directed limited proteolysis.
Limited proteolysis of citrate synthase by Astacus protease, chymotrypsin, clostripain, subtilisin and trypsin on primary fragmentation all yielded similarly sized large (Mr 35 000-36 000) and small fragments (Mr 13 500-14 000) but endoproteinase Lys-C gave fragments of Mr 40 500 and Mr 6500. The sites of the proteolytic attack were determined by Edman degradation of the fragmented synthase preparations, Chymotrypsin, subtilisin, trypsin and endoproteinase Lys-C hydrolyse the synthase at positions 323-324 (-Leu-Arg-), 321-322 (-Ala-Val-)/322-323 (-Val-Leu-), 313-314 (-Arg-Val-) and 366-367 (-Lys-Ala-), respectively. Chymotrypsin and subtilisin attack the small domain of the synthase at the loop between helices O and P very near to a catalytic residue, His-320, and abolish all synthase activities. Primary fragmentation by endoproteinase Lys-C and trypsin reduces the catalytic activity in the physiological overall reaction. Both fragmented enzyme species catalyse the hydrolysis and C-C bond cleavage reactions of citryl-CoA in a stimulated fashion compared to the steady-state rates of the native enzyme, and without hysteretic behaviour. The proteolytic cleavage occurs at acetyl-CoA binding sites within the small domain at the loops connecting helices O to P (trypsin) and Q to R (endoproteinase Lys-C) and reduces the affinity of acetyl-CoA. All of the altered kinetic properties of the fragmented enzyme species are related to this reduced affinity. The correlation between structure and function indicated above is strengthened by the unaltered affinity of oxaloacetate towards the fragmented synthase species. None of the proteolytic enzymes applied attacks oxaloacetate binding sites as defined by the structural work. Oxaloacetate inhibits the hydrolysis of citryl-CoA by the fragmented synthases (endoproteinase Lys-C, trypsin) competitively. An explanation is proposed. The isolated small and large fragments (endoproteinase Lys-C, trypsin) were enzymically inactive. Enzymic activity was restored on recombination of the fragments under denaturing conditions. Cleavage of the loops between helices O to P and Q to R by sequential fragmentation with endoproteinase Lys-C and trypsin inactivated the synthase completely. This result lends support to the idea that the open and closed crystal forms of the structural work are interconverted during the catalytic cycle.
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D002950:Citrate (si)-Synthase; D010450:Endopeptidases; D010447:Peptide Hydrolases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D003546:Cysteine Endopeptidases; C019913:clostripain; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D007652:Oxo-Acid-Lyases