The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The calculation of the rate constants from steady-state kinetics of a single-substrate-single-product enzyme reaction in which there is an isomerization of the enzyme is described. 2. It is shown that even with the use of isotopically labelled substrates a set of solutions for the constants is obtained rather than a unique solution. However, limits are derived within which they must lie. 3. The most appropriate observations to determine the rate constants are measurements of V(max.) and K(m) for both substrate and product, and measurement of the degree of countertransport in an induced-transport test. 4. Experimental procedures for induced-transport tests and the quantitative interpretation of the results obtained are discussed. 5. Product inhibition is shown to be an ambiguous and imprecise means of determining the rate constants. Further, the absence of a [substrate]x[product] term in the denominator of the steady-state rate equation does not necessarily mean that the isomerization of the enzyme is rapid, since the term also disappears when the isomerization is very slow. 6. Similar considerations apply to carrier mechanisms.

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. K , values for ATP, Mg" and K + are greatly increased, the K , for HCO; is not changed much, and the K , for NH: is markedly reduced. V,,, for the synthetase reaction is < 20 % of that of the acetylglutamateactivated enzyme whereas VmaX for the ATPase activity is >40% of that with acetylglutamate. Pulse-chase experiments with H14CO; show formation of less "active C02" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20 -30 pM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATP, (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 pM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time.From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATP, is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the y-phosphoryl group of ATP, appears to be facilitated.It has been known for many years that carbamoylphosphate synthetase of liver mitochondria requires acetylglutamate or an analogue for activity, but the role of this cofactor in the reaction has remained unclear [l]. Recently we have shown that a variety of cryoprotectants' (e.g. dimethylsulphoxide, ethyleneglycol, glycerol and sucrose) partly replace acetylglutamate in activating theenzyme [ 2 ] . The activation by such a range of chemical agents and the lack of evidence for direct participation of acetylglutamate in the enzyme reaction are strong indications that the cofactor and the cryoprotectants activate by an allosteric mechanism. If the mechanism of activation is allosteric the enzyme should possess some activity in the absence of the activator and the kinetics of the nonactivated enzyme should provide information about the activating process. Indeed, although no activity was detected with the frog enzyme [l], about 2 % of the activity has been reported with the rat enzyme in the absence of acetylglutamate' [3]. However, the kinetics of the enzyme in the absence I The term 'cryoprotectants' is applied to various agents that protect cells from the effects of freezing and to those agents that protect cold-labile proteins, such as carbdmoyl-phosphate syntethase 1161. Such compounds are known to stabilize proteins and are frequently added for this purpose rather than to protect specifically against ...

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UMP is a highly specific reagent for photoaffinity labeling of the allosteric inhibitor site of carbamyl phosphate synthetase (CPS) from Escherichia coli and has been found to be photoincorporated in the COOH-terminal domain of the large subunit [Rubio et al. (1991) Biochemistry 30, 1068-1075]. In the present work we identify lysine 992 as the residue that is covalently attached to UMP. This identification is based on two lines of evidence. First, [14C]UMP is found to be incorporated between residues 939 and 1006, as shown by peptide mapping and by mass estimates of [14C]UMP-peptides generated by chemical and enzymatic cleavage of CPS. Secondly, we have purified two radioactive peptides derived exclusively from those enzyme molecules (approximately 5% of the total enzyme) that had incorporated [14C]-UMP. Edman analyses show the sequences of the labeled peptides (989)LVNXVHEGRPHIQD and (989)LVNXVHE to be overlapping. Since neither a phenylthiohydantoin (Pth) derivative (in cycle 4) nor any radioactivity is released from the membrane during sequencing, we can conclude that Lys992 and [14C]-UMP form a covalent adduct that remains bound to the membrane. Formation of this adduct agrees with all of the evidence and with the finding that UMP labeling prevents trypsin cleavage at Lys992. Lysine 992 is invariant in those CPSs that are inhibited by UMP, and is located 30 residues upstream of the site whose phosphorylation in hamster CAD reduces inhibition of CAD by UTP. Multiple sequence alignment of the residues surrounding Lys992 of the E. coli enzyme and the corresponding residues of the yeast and animal enzymes supports the existence of a uridine nucleotide binding fold in this region of the protein. We conclude that sequence changes in the binding fold provide a structural basis for the different regulatory properties found among CPSs I, II, and III.