Changes in the hydrogen exchange kinetics of Escherichia coli ...

Proc. NatL Acad. Sci. USA Vol. 78, No. 11, pp. 6759-6763, November 1981

Biochemistry

Changes in the hydrogen exchange kinetics of Escherichia coli aspartate transcarbamylase produced by effector binding and subunit association (structural fluctuation/allosteric protein /inkage/aspartate carbamoyltransferase)

M. LENNICK AND N. M. ALLEWELL Department of Biology, Wesleyan University, Middletown, Connecticut 06457

Communicated by Frederic M. Richards, July 29, 1981

ABSTRACT Large changes in solvent accessibility to aspartate transcarbamylase (aspartate carbamoyltransferase, carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), as monitored by tritium exchange, result from binding of substrates and substrate analogs to the catalytic subunit (c3), binding of nucleoside triphosphates to the regulatory subunit (r2), and subunit association. Rates of exchange are reduced in each ofthese cases, although to different degrees. Succinate, in the presence of carbamoyl phosphate, retards exchange from c3 no more than carbamoyl phosphate alone, and less than N-phosphonacetyl-L-aspartate, a bisubstrate analog. Larger changes in rates of exchange from r2 are produced by CTP than by ATP; however, both CTP and ATP accelerate exchange from c3 to the same extent. The changes in the kinetics of exchange that result from binding of both substrate analogs and nucleoside triphosphates to the native enzyme (c6r6) are much smaller. Carbamoyl phosphate, with or without succinate, retards exchange only slightly, while the bisubstrate analog has a somewhat larger effect. Experiments with reconstituted enzyme, in which only ct is tritium labeled, indicate that changes in solvent accessibility produced by active site ligands are largely confined to c3. Neither CTP nor ATP alters the overall rate of exchange from c6r6 significantly. The possibility of opposing changes in the two types of subunits was ruled out in experiments in which only one subunit was labeled. The nonadditive effects of ligation and subunit association imply a set of responsive protons common to both processes and suggest that they are linked not only thermodynamically and functionally but also dynamically.

Escherichia coli aspartate transcarbamylase (aspartate carbamoyltransferase, carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is a multimeric protein consisting of three catalytic and two regulatory subunits, designated c3 and r2, respectively, that catalyzes the first committed step in pyrimidine biosynthesis and is subject to feedback regulation by CTP and ATP. (For review, see refs. 1 and 2). Although this enzyme has become an important model system for investigating the molecular basis of allosteric regulation, progress has been slow. A major obstacle has been the difficulty of finding physical probes that have the resolution required by the structural complexity and subtle regulatory mechanism of this enzyme. In the analysis of allosteric systems, the technique of hydrogen exchange has several advantages. Although most spectroscopic probes are able to detect changes only at specific sites in the protein, hydrogen exchange can survey most ofthe structure. Furthermore, the structural fluctuations that determine rates of exchange may play important roles in both catalysis and regulation (cf. refs. 3 and 4). Considerable resolution of discrete The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

kinetic classes can be achieved by using the difference technique developed by Englander and his colleagues (5) and, in favorable cases, the responsive hydrogens can be located in the structure (6, 7, 8). Hence, this method provides a badly needed bridge between structural and thermodynamic studies (cf. ref. 9). Other subunit enzymes that have been characterized by hydrogen exchange include hemoglobin, reviewed by Malin and Eiglander (10), glutamate dehydrogenase (11), pyruvate decarboxylase (12), and chloroplast coupling factor (13). In this investigation, we examined the changes in the kinetics of hydrogen exchange from both the native enzyme (c6r6) and its subunits, (c3 and r2) produced by binding of the major effectors (carbamoyl phosphate, succinate, N-phosphonacetyl-Laspartate (PALA, CTP, and ATP). Because the native enzyme can be readily reconstituted from its subunits, we were also able to determine how ligand binding and subunit association affect individual subunits within c6r6. Our results are novel, in that we find that, in contrast to hemoglobin (10, 14), binding of positive effectors to c6r6 either reduces the rate of exchange or has no effect. Furthermore, the effects of ligand binding and subunit association are not additive. These results are pertinent to current discussions of the mechanisms of hydrogen exchange and allosteric regulation. METHODS PALA was the gift of Robert R. Engle (National Cancer Institute, Bethesda, MD). Its purity was assayed by UV difference spectroscopy as described (15). c6r6, c3, and r2 were prepared according to published procedures (16, 17) from the derepressed diploid strain kindly provided by Howard Schachman (Virus Laboratory, University ofCalifornia, Berkeley, CA). Fermentations were carried out at the New England Enzyme Center (School of Medicine, Tufts University, Boston, MA). The properties ofthe purified proteins, as determined by enzymatic assay and polyacrylamide gel electrophoresis under nondenaturing conditions, were similar to those described (15). For determinations of concentrations of c6r6, c3, and r2, extinction coefficients at 280 nm of 0.59, 0.72, and 0.32 ml-mg-' and Mr values of 310,000, 100,000, and 34,000, respectively (16), were assumed. To facilitate determination of protein concentration in the exchange experiments, trace amounts (