Isolation of Enzymically Active Fragments Formed by Limited Proteolysis of ATP Citrate Lyase

1 Rat-liver ATP citrate lyase was purified to homogeneity and subjected to limited proteolysis by subtilisin, trypsin and chymotrypsin. Each ofthe three proteases yielded two peptides of Mr about 68000 (F-68) and Mr 53000 (F-53) but the subtilisin-derived larger fragment was further degraded to two peptides of Mr 37000 (F-37) and 25000 (F-25). 2 The fragments formed initially had the unimpaired ATP citrate lyase activity and also unimpaired acyl-CoA lyase activity. The former of these but not the latter was destroyed on degradation of fragment F-68 (subtilisin) to fragments F-37 and F-25. 3 Fragments F-53 as formed by all three proteases, and fragments F-70 (chymotrypsin), F-68 (trypsin) and F-(37 + 25) (subtilisin) were isolated chromatographically in homogeneous form. 4 The isolated fragments F-70, F-68 and F-(37 + 25) were characterized as acyl-CoA lyases. The F-53 specimens were enzymically inactive in the applied assays for partial reactions but yielded ATP citrate lyase activity on recombination with fragment F-70 or F-68, not however with fragment F-(37 + 25). 5 From these results and the known position of the catalytic phosphate of ATP citrate lyase it was concluded that limited proteolysis occurs primarily at a region separating acyl-CoA lyase plus part of thiokinase regions from residual thiokinase. 6 A correlation of the sites of catalytic and structural phosphate with distinct functions of the enzyme is presented.