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:Laspartate 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. 1251_ labeled subunits (108 cpm/pg) were used, and the reaction was "stopped" 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 regulatorydeficient molecules and free regulatory subunits gave a second-order rate constant of 105 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-'.Assembly from subunits occurs in seconds at concentrations equivalent to those that would exist in vivo even if there were only one enzyme molecule per cell.Although considerable research has been done on the assembly of large complexes like viruses, ribosomes, flagella and membranes, there have been only a few reports about the sequence of steps in the assembly of the simpler oligomeric enzymes. The allosteric enzyme, aspartate transcarbamoylase; (EC 2.1.3.2; carbamoylphosphate: -aspartate carbamoyltransferase) (ATCase) from Escherichia coli, is ideal for such studies because it is a well-defined oligomer (1) composed of six catalytic (c) and six regulatory (r) polypeptide chains (2-6) and it can be dissociated into catalytic (C) and regulatory (R) subunits and then reconstituted in high yield (1, 7). The c chains are organized as trimers (6) and the r chains as dimers (6,8,9), both in the intact enzyme and after its dissociation with certain mercurials. Recent evidence from electron microscopy (10) and x-ray diffraction studies (11) has provided support for a model of ATCase (6) as a complex of two C subunits bridged by three R subunits, i.e., C2R3. AcAbbreviations: ATCase, aspartate transcarbamoylase; C, catalytic subunit; R, regulatory subunit; c, catalytic polypeptide chain; r, regulatory polypeptide chain; CR3, C2R2, and C2R3, complexes containing C and R subunits in the amounts designated by the subscripts; *C, 125I-labeled C; Cs, succinylated C; CCsR3, an ATCase-like molecule containing one C, one Cs, and three R; TNP, trinitrophenyl. * Submitted in partial fulfillment of the requirements for the Ph.D. degree in Biochemistry.t To whom requests for reprints should be addressed. 3221 cording to this model there are six c: c bonding domains linking the c chains within each of the two trimers, three r: r bonding domai...

Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamoylase

“…In earlier studies on the structure and properties of C2R2 we observed that storage of C2R2 for several weeks in 50 mM Tris.HCI containing 2 mM 2-mercaptoethanol at pH 7.5 led to its partial conversion into a mixture of ATCase and free C subunits (23). Moreover, the disproportionation of C2R2 was more rapid in low ionic strength buffers (5 mM Tris.HCI) and was markedly accelerated upon the addition of PALA (24).…”

“…Very little exchange of either C or R subunits occurred even in the presence of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which promotes the conformational transition of ATCase to the relaxed state (20,21). Studies were conducted, therefore, on the effect of this active-site ligand on the bonding domains in ATCase-like molecules lacking one R subunit (22)(23)(24)(25). This R-deficient species, designated C2R2, which exhibits both cooperativity and inhibition by CTP (22,23), is an intermediate in both the in vitro assembly of ATCase from subunits and the dissociation of the enzyme (24,26 …”

“…Studies were conducted, therefore, on the effect of this active-site ligand on the bonding domains in ATCase-like molecules lacking one R subunit (22)(23)(24)(25). This R-deficient species, designated C2R2, which exhibits both cooperativity and inhibition by CTP (22,23), is an intermediate in both the in vitro assembly of ATCase from subunits and the dissociation of the enzyme (24,26). Since Preparation of ATCase, C2R2, and C and R Subunits.…”

“…About 85% of the *R subunits were converted into ATCase when mixed with excess C2R2. As an additional test of the competence of *R in the reaction mixtures, aliquots were removed and neohydrin was added to a concentration of 6 mM so as to cause the dissociation of the ATCase into C and R subunits (24). After 30 min, 2-mercaptoethanol was added to a concentration of 200 mM.…”

“…The specific activity of labeled C (*C) was 105 cpm/ g and the protein was greater than 95% reconstitutable with an excess of R subunits. 125I-Labeled R (*R) was prepared by reacting R with 125I-labeled methyl-p-hydroxybenzimidate-HCI (24,30). Intact ATCase labeled randomly in both the C and R subunits (*ATCase) with Exchange reactions were performed at 250 with *R at 2.6 MM, ATCase at 0.17 AM, and PALA at 55 MM.…”