©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
L-Aspartate Association Contributes to Rate Limitation and Induction of the T R Transition in Escherichia coli Aspartate Transcarbamoylase
EQUILIBRIUM EXCHANGES AND KINETIC ISOTOPE EFFECTS WITH A V-ENHANCED MUTANT, Asp-236 Ala (*)

Frederick C. Wedler (1), Brenda W. Ley (1), Bong Ho Lee (2)(§), Marion H. O'Leary (2), Evan R. Kantrowitz (3)(¶)

From the (1) Department of Biochemistry and Molecular Biology, Althouse Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802, the (2) Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68583, and the (3) Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Equilibrium isotope exchange kinetics (EIEK) and kinetic isotope effects have been used to determine the mechanistic basis for the altered kinetic characteristics of a mutant version of Escherichia coli aspartate transcarbamoylase in which Asp-236 of the catalytic chain is replaced by alanine (Asp-236 Ala). The [C]Asp N-carbamyl-L-aspartate (CAsp) and [C]CP CAsp exchange rates, observed as a function of various reactant-product pairs, exhibited dramatic increases in maximal rates, along with decreases in substrate half-saturation values and cooperativity. The carbon kinetic isotope effect, C versus C at the carbonyl group of carbamoyl phosphate, for the Asp-236 Ala enzyme decreased toward unity as [Asp] increased, as observed for the wild-type enzyme. Both the kinetic isotope effects and EIEK results indicate that the Asp-236 Ala enzyme operates by the same ordered kinetic mechanism as the wild-type enzyme. Although activation effects by ATP and N-phosphonacetyl-L-aspartate are lost, inhibition by CTP was apparent in equilibrium exchanges. Simulation of the EIEK data indicated that the best fit to the observed changes in saturation curves was obtained by preferentially increasing the rate of the T R transition, k, thereby destabilizing the T-state and increasing the equilibrium constant for the T R transition. A multistep model for Asp binding to aspartate transcarbamoylase is proposed, in which Asp induces the initial conformational changes that in turn trigger the T R transition, followed by stepwise filling of the remaining active sites.


INTRODUCTION

Aspartate transcarbamoylase (EC 2.1.3.2) from Escherichia coli (ATCase)() is the primary regulatory enzyme of the pyrimidine biosynthetic pathway, exhibiting a positive homotropic interaction with the substrate L-aspartate, along with heterotropic activation by ATP and synergistic inhibition by the end products CTP and UTP (1, 2, 3) . The holoenzyme ( M310,000) is composed of catalytic (c) and regulatory (r) subunits with overall stoichiometry cr, arranged as (c)(r)or two trimers of catalytic chains ( M34,000 each) and three dimers of regulatory chains ( M17,000 each). The three-dimensional structure of the enzyme has been determined to high resolution, and structural differences between the T- (low affinity, low activity) and R- (high affinity, high activity) states are well documented (4, 5, 6, 7) . The T- to R-state conversion involves major quaternary structural changes, including a 12-Å expansion along the 3-fold molecular axis, and rotation of the regulatory subunits about their respective 2-fold axes (8) , along with more subtle tertiary structural changes. These include closure of the cleft between the two domains of each catalytic chain, and adjustments of the structure within the Asp binding site. In addition, within each catalytic chain, a section composed of residues 230-250 (the 240s loop) undergoes a major reorientation. For the T-state holoenzyme, specific side chain interactions also occur between catalytic chains of the two catalytic trimers, and between Asp-236 of the catalytic chains and Lys-143 of the regulatory chains (9) . Analysis of the Asp-236 Ala()enzyme has revealed the importance of these interactions for both homotropic and heterotropic interactions in the enzyme (10) . Initial velocity kinetics indicated that the mutant holoenzyme lacked cooperativity for substrate binding, while substrate affinities have increased by 3-10-fold. Most striking was an increase in Vof 25%. Only 2 or 3 of the 50 or more site-specific mutants of ATCase prepared as of 1990 exhibit such increases in the maximal velocity (11) . Mutation of Asp-236 Ala and Tyr-240 Phe (10, 12) , both in the 240s loop, are almost unique in causing a significant increase in V.

One of the major unanswered questions about the dynamic behavior of ATCase regards the rate-limiting step in the forward direction. The properties of the Asp-236 Ala enzyme make it valuable for gaining new insights toward this question. An in-depth kinetic analysis is required to define changes in individual rate constants. Equilibrium isotope exchange kinetics (EIEK) and kinetic isotope effects (KIE) have proven highly insightful with ATCase (13, 14, 15, 16, 17, 18, 19) . Unique to EIEK methods is the ability to observe simultaneously both the fast and slow steps for substrate association-dissociation in both directions, involving binary and ternary complexes. For example, it was found that the allosteric modifiers ATP and CTP do not perturb the T R transition directly; instead, they differentially alter the Asp association rate more than the dissociation rate (20) . For the Tyr-240 Phe mutant, computer simulations of EIEK data elucidated changes in the kinetic mechanism (13, 14) . KIE methods have been shown to be sensitive to alterations in the chemical and kinetic mechanism of the wild-type and mutant enzymes (13, 14, 15, 16, 17, 18, 19) . The present investigation involves using both these methods to investigate kinetic changes caused by the Asp-236 Ala mutation, which are relevant for helping to define in greater detail the rate-limiting step in the forward direction for the wild-type enzyme.


EXPERIMENTAL PROCEDURES

Materials

Biochemicals were of the highest purity available from Sigma. Other chemicals were ACS reagent grade (Fisher Scientific). Carbamyl phosphate (dilithium salt) was purified prior to use by precipitation from 50% (v/v) ethanol:water and was stored desiccated at 20 °C. [C]L-Asp (Amersham Corp., 50 µCi, 216 mCi/mmol) was purified from radiolytic contaminants prior to use by application of the entire sample to a 0.3 1.0-cm column of Dowex-50 AG-X8 (H, 100-200 mesh), followed by a 15-ml water wash, then elution with 10 ml of 2 M HCl. After taking the eluant to dryness twice by rotary evaporation, the residue was dissolved in 1 ml of 20% ethanol:water and stored at 20 °C. [C]Carbamyl phosphate (DuPont NEN, 50 µCi, 17.6 mCi/mmol) was dissolved in 10 ml of water, divided into 100-µl aliquots, flash-frozen with liquid nitrogen, and stored at 80 °C. These aliquots of labeled CP were thawed and used individually and the residual material was flash-frozen no more than once to avoid breakdown to cyanate.

Enzymes

Wild-type ATCase was prepared as described by Nowlan and Kantrowitz (21) from E. coli strain EK1104 containing the plasmid pEK2. The Asp-236 Ala ATCase was prepared as described previously (10) .

General Methods

Enzyme activity was assayed using either the formation of [C]CAsp from labeled Asp (22) , a continuous spectrophotometric coupled phospholysis method (23) , or a pH indicator assay.() One unit of activity is defined as the number of micromoles of product (CAsp) formed per minute. Protein concentration was determined by the Lowry method (24) after trichloroacetic acid precipitation, by the bicinchoninic acid (BCA) method (Pierce), or by absorbance measurements at 280 nm with an extinction coefficient of 0.59 cm/mg (25) .

Equilibrium Isotope Exchange Kinetics

Equilibrium isotope exchange experiments were carried out according to established procedures, described in detail elsewhere (26, 27, 28) . The concentrations of substrate-product pairs were varied from well below to well above their Kvalues as follows; solution A (containing reaction components at twice their final concentrations) was mixed in different proportions to the same final volume (0.1 ml) with solution B (lacking the varied components). After thermal equilibration for 5 min at 30 °C, 5 µl of enzyme was added and the mixture was incubated for 15 min to allow for exact catalytic adjustment to chemical equilibrium. Isotopic exchange was then initiated by addition of labeled substrate, [C]L-Asp (0.1 µCi) or CP (0.02 µCi), in micromolar quantities less than 1/1000 of the material in the unlabeled pool so as to avoid perturbation of the equilibrium condition. Exchange reactions were typically carried out for 20 min at 30 °C, then quenched and separated for counting.

[C]Asp C-Asp exchange reactions were quenched by addition of 0.45 ml of 0.02 M HCl and chilling on ice prior to separation of the labeled pools, which was carried out exactly as described above for the purification of [C]Asp, using a 0.3 5-cm column of Dowex 50 (H). The [C]CP CAsp exchange reactions, carried out in 1.5-ml Eppendorf tubes, were quenched by addition of 0.15 ml of 5 N HCl. The tubes were closed, the caps pierced, and the [C]COremoved in vacuo (<0.5 mm Hg) for 75 min, using a Savant SpeedVac concentrator with a refrigerated trap plus an Ascarite (5 20 cm) trap prior to the vacuum source.

Calculations

For exchange of isotopic label at chemical equilibrium, X* Y, the number of micromoles exchanged/min ( R) was calculated according to Equation 1 (29) , where X and Y represent the micromoles of substrates present in a given reaction, t = time (min), and F is the fraction of isotopic equilibrium attained, equal to y( X + Y)/( x + y) Y, where x and y are the disintegrations/min values observed in the X and Y pools, respectively.

 

On-line formulae not verified for accuracy

EIEK Data Analysis

The shapes of saturation curves in EIEK experiments distinguish between compulsory and random order sequential kinetic mechanisms (26, 27, 28) . EIEK methods are unique in their ability to allow simultaneous observation of both the rate-limiting and non-rate-limiting steps in both directions, which include the formation and breakdown of both the ``inner'' and ``outer'' complexes. A newly developed mouse-interactive calculational program, ISOBI-HS, was used to fit entire sets of equilibrium isotope exchange data (28) . These procedures lead to an optimal set of rate constants, as described elsewhere in theory and practice (13, 14, 20, 28) . The rate constants must meet several other criteria in order to be considered valid. Specifically, within determined confidence limits (allowed limits of variation) they must produce chemical equilibrium for all closed cycles and predict the correct initial velocities in both directions, using the KINSIM program (30) . The kinetic constants and saturation curves for the mutant are then compared to those for wild-type enzyme, thereby determining the specific rate constants altered by the mutation. The theoretical curves in the figures were generated using the best fit parameters from the ISOBI-HS program (28) .

Isotope Effects

Isotope effects are described using the terminology of Northrop (31) . Thus, (V/K)represents the observed kinetic isotope effect for C versus C at the carbonyl group of CP. (V/K)on the ATCase reaction was measured using the method of internal competition, in which changes in the isotopic composition of CP are measured over the course of the reaction (17) . A 200 mM CP solution, pH 5.5, was prepared in water, sealed with a rubber septum, placed in an ice bath, and freed from COby sparging with Nfor 1-2 h. A reaction flask fitted with a vacuum adapter and a side arm was charged with an appropriate amount of 50 mM HEPES, pH 7.5, containing 2 mM dithiothreitol and 0.2 mM EDTA, and this solution was sparged overnight with N. A 100 mM Asp solution was prepared in this same buffer, sealed with a septum, and sparged with Novernight at room temperature. 50 mM ATP and CTP solutions were prepared in 50 mM HEPES containing 2 mM dithiothreitol and 0.2 mM EDTA, pH 7.5, and were likewise sparged. To the reaction flask containing HEPES buffer were added appropriate amounts of the aspartate solution and ATCase. The reaction was initiated by addition of 1 ml of ice-cold CP solution. At low Asp concentrations, in order to keep the Asp concentration constant, small amounts of degassed Asp solution were added dropwise at a rate calculated to keep the Asp concentration at the desired level. After a reaction time estimated to attain 50% reaction, 0.5 ml of concentrated sulfuric acid was added to the reaction mixture via a syringe and the solution was warmed to 38 °C for 2 h to convert unreacted CP to CO(>10 half-lives of CP hydrolysis; at pH 0.35, 37 °C, the half-life for CP is 11.3 min (32) . The resulting COwas collected by a high vacuum continuous distillation apparatus using two dry ice/isopropanol traps and a liquid nitrogen trap. The trapped COwas further purified by bulb to bulb distillations. The amount of COproduced was measured manometrically using a MKS PDR-D1 READOUT pressure gauge calibrated with acidified potassium carbonate solution. The isotopic content of the resulting COwas measured by isotope ratio mass spectrometry on a Finnigan Delta-S isotope-ratio mass spectrometer equipped with a Heraeus combustion unit.

Two methods were used to determine the isotopic content of unreacted CP. In the first, CP was converted to COby acid treatment. 1 ml of sparged CP solution in water was added to a flask containing 15 ml of HEPES buffer solution. Acidification of this solution with 0.5 ml of concentrated sulfuric acid resulted, after 2.5 h at 38 °C, in the decomposition of all the CP to give CO, which was isolated and analyzed as described above. Alternatively, combustion analysis was used to obtain the isotopic content of CP. The two methods gave the same results, within experimental error.


RESULTS

Equilibrium Isotope Exchange Kinetics

Saturation curves for the [C]Asp CAsp and [C]CP CAsp exchanges, catalyzed by the Asp-236 Ala holoenzyme at pH 7.0, 30 °C, resulting from variation of both reactants and both products in constant ratio at equilibrium, are shown in Fig. 1. For comparison, the curves obtained for wild-type holoenzyme under identical conditions (29) are indicated by dotted lines. The shapes of the curves are altered in a manner very similar to the changes observed by initial velocity kinetics upon variation of the concentration of Asp (10) . Specifically, the half-saturation value decreased markedly, and the maximal rate increased. Whereas Vincreased only about 20% (10) , the observed increase in Rfor isotope exchange is increased by 2-fold for Asp CAsp and by almost 3-fold for CP CAsp. The strong inhibition effects for CP CAsp, due to ordered substrate binding (10) , are even more apparent for the Asp-236 Ala holoenzyme (Fig. 1), along with weak inhibition effects observed previously for Asp CAsp. These latter observations suggest that the Asp-236 Ala holoenzyme operates by a kinetic mechanism essentially identical to that for the wild-type holoenzyme, namely CP binds prior to Asp and CAsp is released prior to P. The data in the next several figures serve to verify this hypothesis.


Figure 1: The effect of varying the concentrations of all substrates in constant ratio on the rates of the [C]Asp CAsp and [C]CP CAsp exchange reactions, catalyzed at chemical equilibrium by the Asp-236 Ala enzyme at pH 7.0, 30 °C. The maximum concentrations of substrates at f = 1.0 were (in mM): CP (0.185), Asp (185), P(436), and CAsp (463). Each reaction also contained 100 mM PIPES buffer and 100 mM KCl. Dotted line, saturation curve for the wild-type enzyme (30).



Fig. 2 shows the effects on the Asp CAsp and CP CAsp exchange rates of varying all possible reactant/product pairs in constant ratio, catalyzed by the Asp-236 Ala holoenzyme. Variation of the Asp/CAsp pair (Fig. 2 A) causes Asp CAsp to rise smoothly to a maximum, but results in sharp peaking and inhibition of the CP CAsp exchange. In contrast, variation of the CP/Ppair (Fig. 2 B) simply caused both exchanges to rise smoothly to a maximum, as expected for the preferred order binding mechanism described above. Weak substrate inhibition effects seen for the wild-type are absent for the Asp-236 Ala holoenzyme. Strong enhancement of the maximal rate for the CP CAsp exchange is even more apparent in Fig. 2than in Fig. 1, with almost 5-fold increases for the Asp-236 Ala enzyme compared to the wild-type holoenzyme. In both experiments, both exchanges showed markedly decreased half-saturation values as well.


Figure 2: Rates of the [C]Asp CAsp and [C]CP CAsp exchange reactions as a function of the concentrations of reactant-product pairs, varied in constant ratio at chemical equilibrium, catalyzed by the Asp-236 Ala mutant of ATCase at pH 7.0, 30 °C. Each reaction also contained 100 mM PIPES buffer and 100 mM KCl. The concentrations of the nonvaried pairs were held constant corresponding to f = 0.4 in Fig. 1. Variations are as shown: A, Asp and CAsp, holding [CP] and [P] constant at 0.074 and 174 mM, respectively; B, CP and P, holding [Asp] and [CAsp] constant at 74 and 185 mM, respectively; C, Asp and P, holding [CP] and [CAsp] constant at 0.074 and 185 mM, respectively; D, CP and CAsp, holding [Asp] and [P] constant at 74 and 174 mM, respectively. Dotted lines, saturation curves for the wild-type enzyme (30).



Variation of Asp/P(Fig. 2 C) results in strong inhibition of the CP CAsp exchange rate, with weaker effects on the Asp CAsp exchange. The fact that weak inhibition of the Asp CAsp exchange was not observed in Fig. 2 A for the variation of Asp/CAsp suggests that this effect is due to dead-end complex formation, perhaps EPAsp. Similar weak inhibition effects are seen in Fig. 2 D upon variation of CP/CAsp. By similar logic, comparison of these curves to those in Fig. 2 B suggests that CAsp also forms a dead-end complex, probably ECAspCP. The data in Fig. 2 ( C and D) clearly show a stronger increase in maximal rate for the CP CAsp exchange, compared to Asp CAsp. Overall, the data in Fig. 2verify that the Asp-236 Ala holoenzyme operates by a kinetic mechanism that is essentially identical to that for the wild-type holoenzyme, namely a nearly compulsory order scheme in the forward (17, 29) and reverse (29) directions. Inhibition effects that are diagnostic of this scheme are even more distinct with the Asp-236 Ala enzyme than with the wild-type holoenzyme.

Kinetic Isotope Effects

The C isotope effect, measured for the reaction of [C]CP with Asp, catalyzed by the Asp-236 Ala enzyme as a function of the concentration of Asp in the presence of a fixed and saturating concentration of CP, is shown in Fig. 3. The shape of the saturation curve, defined by the data for the wild-type holoenzyme, is shown as a dotted line (29) . The data for the mutant enzyme are identical to those for the wild-type enzyme, within experimental error.


Figure 3: Kinetic isotope effect, (V/K), for the forward reaction catalyzed by the Asp-236 Ala mutant of ATCase as a function of [Asp] in the presence of fixed [CP] (20 mM), pH 7.5, 25 °C. Each reaction also contained 50 mM HEPES buffer, 2 mMdithiothreitol, and 0.2 mM EDTA. Dotted line, saturation curve for the wild-type enzyme (17).



The fundamental conclusion derived from the observed decrease in (V/K)from 1.025 at low [Asp] to unity at saturating [Asp] is that the kinetic mechanism is ordered. If the kinetic mechanism were random, the isotope effect would remain finite and constant at high [Asp], since CP could escape from the central complex, ECPAsp. In the case of a compulsory order mechanism, however, although at low [Asp] bound CP can freely dissociate and isotopic discrimination occurs, as [Asp] increases bound CP is increasingly prevented from dissociating by the rapid binding of the second substrate, Asp (which results immediately in a high commitment to catalysis). Thus, the observed kinetic isotope effects are in accord with the ordered kinetic mechanism determined from the isotope exchange data in Figs. 1 and 2.

Effector Ligands

By initial velocity kinetics, the Asp-236 Ala enzyme appears to behave as a nearly R-state form (10) , based on the almost 10-fold decrease in Sand loss of cooperative binding for Asp, and simultaneous loss of activation effects by the bisubstrate analog, PALA. With wild-type holoenzyme under conditions of [Asp] below S, low concentrations of PALA (<1 µM) cause activation, due to sub-stoichiometric amounts of bound PALA converting the low activity, low affinity T-state enzyme to the high activity, high affinity R-state. Lack of PALA activation indicates that the Asp-236 Ala enzyme is already in the R-state, even at saturating CP and low [Asp]. The isotope exchange data in Fig. 4fail to detect any PALA activation of either Asp CAsp or CP CAsp, even at concentrations near 1 µM. Inhibition effects are due to direct competition of PALA with substrates.


Figure 4: Effects of varying the concentration of the bisubstrate analog, PALA, on the rates of the [C]Asp CAsp and [C]CP CAsp exchanges at chemical equilibrium, catalyzed by Asp-236 Ala enzyme at pH 7.0, 30 °C. Substrate concentrations were held constant at concentrations corresponding to f = 0.15 in Fig. 1.



Another notable feature of the Asp-236 Ala enzyme was loss of sensitivity to modifiers, ATP and CTP under initial velocity conditions (10) . Using EIEK methods, however, these modifiers alter the Asp CAsp and CP CAsp saturation curves (as a function of various reactant/product pairs), as shown in Fig. 5. The Asp-236 Ala enzyme is insensitive to ATP (except for weak inhibition of CP CAsp upon variation of the CP/Ppair), but in all cases CTP caused strong, differential inhibition of the CP CAsp exchange, but not the Asp CAsp exchange. Furthermore, CTP alters the Rbut not the half-saturation value for the CP CAsp exchange, as also occurred with the wild-type enzyme (20) . A similar differential loss of modifier sensitivity was observed with the pAR5 enzyme described by Hervé, Cunin, and co-workers (33) . Differential changes in modifier sensitivity can provide important clues about which kinetic steps have been altered by the mutation, as will be discussed below.


Figure 5: Effect of modulators (M) on the exchange rates at chemical equilibrium, catalyzed by the Asp-236 Ala ATCase enzyme, pH 7.0, 30 °C. Data are shown for 0.5 mM CTP and 2 mM ATP on the [C]Asp CAsp and [C]CP CAsp exchanges. The concentrations of substrates, varied together in constant ratio, and other components were as in Figs. 1-3. Concentrations of nonvaried components in the two bottom panels corresponded to f = 0.4 in Fig. 1.



Data Analysis

A useful starting point for analyzing the effects of the Asp-236 Ala mutation is to consider the kinetic properties of previously studied forms of ATCase that exhibit ``activation,'' i.e. increased Vand decreased Svalues. These enzyme forms include the catalytic subunit of the wild-type enzyme (20) , the ATP-ligated holoenzyme (20) , and two R-like mutants, Tyr-240 Phe (13) and Glu-239 Gln (34) . The properties of these forms are summarized in . The first impression from this summary is that the Asp-236 Ala enzyme appears to most closely resemble the wild-type catalytic subunit in its properties.

This apparent similarity is not supported by a more detailed comparison, however. First, considering kinetic isotope effects, the saturation curve for (V/K)for wild-type catalytic subunit does not go to unity (17) and therefore maintains a degree of random character not seen with the Asp-236 Ala or wild-type holoenzymes, although the data for the Asp-236 Ala enzymes do not rule out a very small random component to the mechanism. Second, from equilibrium isotope exchange kinetics, distinctive differences in the kinetic parameters Rand Sfor the Asp CAsp and CP CAsp exchanges are observed for wild-type catalytic subunit and the Asp-236 Ala holoenzyme; these parameters are listed in , along with those for the wild-type holoenzyme.

By comparing how the kinetic parameters for each of these forms differ from those for wild-type holoenzyme, a set of distinctive patterns emerges. In order to analyze these effects systematically, the perturbations in Rand Scompared to the wild-type enzyme were calculated and plotted in bar graph form, as shown in Fig. 6(see ). This method of comparison has proven highly effective in defining which steps are altered by bound feedback modifiers (20) , as well as by site-specific mutations (13, 34) .


Figure 6: Perturbations in EIEK parameters of ``activated'' forms of ATCase, compared to wild-type holoenzyme, pH 7.0, 30 °C, calculated from data in Table II (see text). Stippled bar, V(µmol/min/mg); shaded bar), 1/ S. The enzyme forms are as follows: A, wild-type catalytic subunits; B, wild-type ATP-liganded holoenzyme; C, Asp-236 Ala holoenzyme; D, Tyr-240 Phe holoenzyme; E, Glu-239 Gln holoenzyme.



Although it is clear that the kinetic behavior of the Asp-236 Ala enzyme is unique, this comparison indicates a close resemblance of this mutant to both the wild-type holoenzyme with ATP bound and the Glu-239 Gln enzyme. Although several other ``activated'' forms exhibit increases in substrate affinity (1/ S) values comparable to those observed for the Asp-236 Ala enzyme, they also exhibit decreases in R, whereas the Asp-236 Ala enzyme shows distinctive increases in Rfor both exchanges. The match in perturbation of parameters (Fig. 6) is best for ATP-ligated wild-type and Glu-239 Gln enzymes in experiments involving variation of the concentrations of all substrates or the Asp/CAsp pair. When the CP/Ppair was varied, only the wild-type holoenzyme with ATP bound exhibited perturbations that resembled those observed for the Asp-236 Ala enzyme.

Using these initial clues as a working hypothesis, simulation of the kinetic data for Asp-236 Ala enzyme was carried out with the ISOBI-HS program. First, the effect of altering the association and dissociation rates for Asp and CAsp was determined, both in equal ratio and by differentially increasing k> k. These changes, as observed previously (20) , enhanced Rand decreased Sfor Asp CAsp, but had the opposite effect on the CP CAsp exchange. This is not observed with the Asp-236 Ala enzyme, for which both exchanges exhibit increased exchange rate and substrate affinities (decreased S). The only changes that produced the exact effects observed with the Asp-236 Ala enzyme in Figs. 1 and 2 were a decrease in the Hill number ( n) from 2.2 to 1.5 plus slightly slower release of Asp from two dead-end complexes, EPAsp and ECAspAsp.


DISCUSSION

Disrupting the interchain interaction between Asp-236 of a catalytic chain and Lys-143 of the regulatory chain destabilizes the T-state of ATCase (10) . The current kinetic results, in conjunction with structural considerations, are consistent with the mutation causing an increase in k(but not k) for the T R transition, which increases the allosteric parameter L k/ k. In accordance with these findings, low angle x-ray scattering data show that unliganded Asp-236 Ala enzyme has a quaternary conformation that is neither T nor R, but that binding of CP alone converts the Asp-236 Ala enzyme 75% toward the quaternary conformation observed for wild-type R-state.()The kinetic data in Figs. 1-3 also clearly show that the Asp-236 Ala substitution does not alter significantly the ordered kinetic mechanism observed for wild-type holoenzyme.

Of particular significance for discerning which steps contribute to overall rate-limitation, the maximum activity for the Asp-236 Ala enzyme is almost twice that of wild-type enzyme by both EIEK and initial velocity methods.()Comparison of EIEK saturation curves for the Asp-236 Ala enzyme to those for various ``activated'' forms of ATCase indicate that the Asp-236 Ala enzyme most closely resembles the ATP-liganded wild-type enzyme, which has substantial R-state character (20) . In accordance with this analysis, x-ray crystallographic data indicate that the binding of ATP specifically disrupts the link between Asp-236 (catalytic) and Lys-143 (regulatory) (35) .

These and previous findings suggest that binding of Asp to ATCase is a complex, multistep process. Overall, this involves both gross quaternary structural changes (the T R transition) as well as more subtle secondary structural changes associated with stepwise binding to the active site once a state with appreciable affinity for Asp has been achieved. These different levels of structural changes have been the subject of hypothesis and experimentation by a number of researchers in recent years, including Tauc et al. (36) . Clearly, the simple two-state M-W-C (concerted) model (2) for cooperative substrate binding is an oversimplification for this system. An attempt to incorporate these structural changes into a model that relates them to the observed kinetics and mechanism is outlined in Schemes I and II.

A key feature of the mechanism for Asp cooperativity depicted in Schemes I and II is ``induction'' of the initial conformational change caused by the binding of Asp to the low affinity form of ATCase. Based on x-ray structural data (35) , binding of CP alone alters the free T-state enzyme. CP-liganded enzyme is designated T` to distinguish it from the pure T-state (unligated), which cannot bind Asp. The first molecule of Asp to bind triggers ( induces) formation of an activated, highly transient T-state form, designated T*, which rapidly undergoes concerted conversion of all three chains to a near-R-state conformation designated R`. This is in accord with the observation that only one PALA molecule per catalytic trimer results in a complete T R transition (37) . The structural basis for expansion of quaternary structure along the 3-fold axis by 12 Å and movement of the 240s loops of the catalytic chains past each other has been described in detail (38) . Once R` is formed, stepwise filling of the additional sites, driven by mass action of [Asp], produces the fully active R-state enzyme.

At a more detailed structural level, Fig. SIIindicates that binding of the first Asp (in step 1) triggers disruption of specific bonds that stabilize the T`-state. In the current model, these are suggested to be residues in the 240s loop such as the intrachain hydrogen bonds and salt links between Tyr-240-Asp-271 and Arg-229-Glu-272. Binding of the first Asp produces the labile, activated state T*, the least stable intermediate in this process, which readily converts its gross, quaternary structure to R`. This latter form is proposed to have enhanced affinity for Asp without having achieved an active site conformation with full catalytic activity. Attaining the fully active R-state depends on stepwise binding of Asp to the remaining active sites (in step 2) by mass action. As described above, this drives completion of domain closure and brings active site groups into proper juxtaposition, to facilitate the ``compression'' mechanism (39) in which the carbonyl carbon of CP undergoes nucleophilic attack by the -amino group of Asp.


Figure SII: Mechanistic hypothesis for stepwise substrate binding to ATCase, coupled to gross quaternary (T R) and more subtle secondary structural conformational changes.



The kinetic isotope effects observed by Parmentier et al. (17) indicated that catalysis contributes roughly 50% to rate limitation, with a ``precatalytic step'' determining the remainder. The question to be addressed now is: which of the steps in Fig. SII contributes most significantly to rate limitation? As discussed by Ray (40) , the concept of rate limitation for enzyme-catalyzed reactions is complex. Knowles and Albery (41) have espoused the view that there is no evolutionary reason for enzymes to evolve such that any single step in the catalyzed reaction is much faster than any other. In fact, EIEK and related kinetic methods (28) indicate that catalysis is rate-limiting for only a few enzymes.

Data in the current literature allow us to eliminate certain steps in Fig. SIIas candidates for the non-catalytic rate-limiting step, specifically step 1 and the T R transition. Converting ATCase to forms that are more R-like (either in quaternary structure or kinetically or thermodynamically) does not enhance the maximal rate. Examples of these include the wild-type catalytic subunit, as well as the Tyr-240 Phe and Glu-239 Gln holoenzymes (11, 13, 34) , which are R-like on the basis of decreased S(Asp) but for which Vis not significantly different from wild-type holoenzyme, after correcting for substrate inhibition. In addition, it is significant that ligand-free enzyme and enzyme with ATP and CTP bound all exhibit the same initial value of (V/K)at low Asp, as well as identical hyperbolic Asp saturation curves for (V/K). This indicates that all three operate via the same enzyme form (17) , which argues that step 1 and the T R transition are relatively rapid, compared to step 2. This leaves step 2 in Fig. SIIas the prime candidate for rate limitation.

CTP, which clearly pushes the T R equilibrium toward the T-state, does not alter V. In contrast, ATP does cause an increase in V, which is particularly evident by EIEK methods (20) and after correction for substrate inhibition effects. These differences suggest that CTP alters a step in Fig. SIIthat does not contribute to rate limitation, whereas ATP alters a step that does. Furthermore, these differences indicate that at a detailed structural level, ATP and CTP alter the properties of ATCase by independent mechanisms (20) , even though at a macroscopic level they simply perturb the T R equilibrium in opposite directions.

  
Table: Summary of kinetic properties of wild-type and Asp-236 Ala enzymes


  
Table: Kinetic properties of activated forms of ATCase, compared to the Asp-236 Ala mutant, determined by equilibrium isotope exchange kinetics upon variation of different combinations of reactant-product pairs in constant ratio, pH 7.0, 30 °C



FOOTNOTES

*
This work was supported by National Science Foundation Grants MCB89-03546 and MCB93-19035 (to F. C. W.), National Institutes of Health Grant GM43043 (to M. H. O.), and National Institutes of Health Grant GM26237 (to E. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Dr. Frederick C. Wedler, who died on December 1, 1994. During his career he made important contributions to the understanding of a variety of enzymes including aspartate transcarbamoylase using equilibrium isotope exchange kinetics.

§
Current address: Dept. of Industrial Chemistry, Taejon National University of Technology, Samsung 2-dong, Dong-ku 300-172, Korea.

To whom correspondence should be addressed.

The abbreviations used are: ATCase or cr, aspartate transcarbamoylase holoenzyme; c, catalytic subunit of aspartate transcarbamoylase; CP, carbamyl phosphate; Asp, L-aspartate; CAsp, N-carbamyl-L-aspartate; PALA, N-phosphonacetyl-L-aspartate; PIPES, piperazine- N, N`-bis-[2-ethanesulfonic acid]; EIEK, equilibrium isotope exchange kinetics; KIE, kinetic isotope effects.

The notation used to name the mutant enzymes is shown, for example, by the Asp-236 Ala enzyme. The wild-type amino acid and location within the catalytic chain is indicated to the left of the arrow, while the new amino acid is indicated to the right of the arrow.

B. H. Lee and M. H. O'Leary, unpublished data.

P. Vachette, personal communication.

Reanalysis of the data of Newton & Kantrowitz (10) indicated that Asp-236 Ala enzyme binds Asp with weak cooperativity. Lineweaver-Burk and Eadie-Hofstee plots were concave, and Hill plots gave n= 1.4-1.5. Nonlinear fitting of the data according to an equation similar to that of Pastra-Landis et al. (42) to correct for substrate inhibition effects indicated V 28-32 mmol/h/mg.


ACKNOWLEDGEMENTS

We thank Dr. P. Vachette for making available the results of low angle x-ray experiments prior to publication. We also thank Dr. S. C. Pastra-Landis and Dr. D. Baker for critically reviewing this manuscript.


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