title: Pathways of assembly of aspartate transcarbamoylase from catalytic and regulatory subunits.
abstract: A scheme for the assembly of aspartate transcarbamoylase (EC 2.1.3.2; carbamoylphosphate:L-aspartate carbamoyltransferase) from catalytic and regulatory subunits is presented along with a technique for detecting intermediates and measuring the kinetics of assembly at protein concentrations of about 10 nM. (125)I-labeled subunits (10(6) cpm/mug) were used, and the reaction was "topped"at specific times with an appropriate "chase" followed by electrophoretic separation and measurement of the amounts of the various species. Two intermediates were identified. A stable enzyme complex lacking one regulatory subunit is the principal product when catalytic subunits are in excess. A transiently stable complex lacking one catalytic subunit is the principal species when regulatory subunits are in excess. Measurements with mixtures of the purified regulatory-deficient molecules and free regulatory subunits gave a second-order rate constant of 10(5) 10(5) M(-1) sec(-1) for the formation of bonding domains between catalytic and regulatory chains. No data for the rate of rupture of these bonds are available, but the kinetics of the assembly of the enzyme in vitro can be accounted for if this value is about 10(-2) sec(-1). Assembly from subunits occurs in seconds at concentrations equivalent to those that would exist in vitro even if there were only enzyme molecule per cell.
mesh_terms: D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000378:metabolism; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D007457:Iodine Radioisotopes; D010455:Peptides; D001221:Aspartate Carbamoyltransferase