Modes of modifier action in E. coli aspartate transcarbamylase

ARCHIVES

OF

Modes

BIOCHEMISTRY

AND

of Modifier

BIOPHYSICS

Action

FREDERICK Chemistry

Department,

Cogswell

163,

69-78 (19%)

in E. coli Aspartate

C. WEDLER’ Laboratory, Received

AND

Rensselaer December

FRANK Polytechnic

Transcarbamylase J. GASSER Institute,

Troy,

New

York

16181

20, 1973

The observed patterns for inhibition by CTP and succinate of equilibrium exchange kinetics with native aspartate transcarbamylase (E. coli) are consistent with an ordered substrate-binding system in which aspartate binds after carbamyl phosphate, and phosphate is released after carbamyl aepartate. ATP selectively stimulates Asp % carbamyl-Asp exchange, but not carbamyl phosphate + Pi. Initial velocity studies at 5”, 15”, and 35°C were carried out, using modifiers as perturbants of the system. Modifiers alter the Hill n and So.5 for sspartate, most markedly at 15°C but less so at the other temperatures. ATP does increase I’ under saturating substrate conditions, and substrate inhibition is observed for aspartate. ATP does not make the Hill n = 1 at any temperature. It is proposed that CTP and ATP act by separate mechanisms, not by simply perturbing in opposite directions the equilibrium for aspartate binding. ATP appears to act to increase the rate of aspartate association and dissociation, whereas CTP induces an intramolecular competitive effect in the protein.

The exact modes of action of the modifiers CTP and ATP with E. coli aspartate transcarbamylase have been the subjects of intense interest and research in recent years (1). These heterotropic effects, in relation to the homotropic substrate binding processes, have been studied by a variety of techniques, especially in terms of the protein conformational changes associated with them. These include equilibrium binding (2, 3), difference uv-spectroscopy (4, 5), proteolytic digestion (6-S), ultracentrifugal sedimentation velocity and equilibrium studies (9, lo), thiol group reactivity (11, 12), circular dichroism (13), T-jump relaxation spectra (1417), initial velocity kinetics (1, l&20), transient nmr (21-23), and fluorescence (24, 25) approaches. Interestingly, genetic mutation of the regulatory subunit, suggesting disconnection of the homotropic (substrate-induced) and heterotropic (modifier-induced) changes, has been reported recently (26). From these studies have arisen a variety of working hypotheses and models for regulation of the enzyme i To whom correspondence

(10, 27-30). Clearly, other means are required for distinguishing among these. Whereas X-ray crystallographic studies now in progress (30) will resolve certain points in question for static structures or conformations, additional dynamic probes are also needed. Equilibrium isotopic exchange kinetics (31, 32) have proved to offer a unique and incisive means of defining modifier action and other aspects of enzyme mechanisms. For example, a study of the interaction of E. coli glutamine synthetase with some eight or nine feedback modifiers (33, 34) has yielded new insight to modifier action. This study included the derivation of theoretical inhibitory patterns for alteration of particular microscopic kinetic steps in a two-reactant, two-product? enzyme systems, with either random or ordered substrate binding (35). Such patterns and other diagnostic parameters have been used here * Nonstandard abbreviations used in text of this paper include: Asp, L-aspartic acid; C-Asp, N-carbamyl-L-aspartate; C-P, carbamyl phosphate; ATCase, aspartate transcarbamylase; CSU, catalytic subunits of ATCase; and Vi, initial velocity.

should be addressed. 69

Copyright All rights

0 1974 by Academic Press, of reproduction in any form

Inc. reserved.

70

WEDLER

AND

to analyze the effects of CTP, ATP, and succinate on native ATCase, and provide further evidence for ordered substrate binding at 30°C (36, 37). In addition, the basis for the nonlinear temperature dependence of net catalysis (36) is probed by both equilibrium exchange and initial velocity kinetics, using modifiers as perturbants. This has allowed further dissection and analysis of the steps involved in catalysis and allosteric regulation. EXPERIMENTAL

SECTION

The materials and procedures used in these experiments have, for the most part, been described previously in the accompanying paper (36). This includes the preparation and storage of aspartate transcarbamylase from E. coli, the separation of labeled Asp and C-Asp, or C-P and Pi,‘equilibrium exchange procedures, and the initial velocity pHstat technique and equipment. Substrates, succinate, and nucleotides were products of Sigma Chemical Co., tested for purity, and used without further purification. Labeled materials, 3PPi and [“C]Asp, were products of New England Nuclear. Variation of modifier levels without perturbation of pH, ionic strength, substrate levels, or other conditions in equilibrium experiments was accomplished as follows: two solutions, A and B were prepared, one lacking but the other containing modifier, but identical in pH and substrate levels and with relatively high ionic strength (0.2 M) achieved with added KCl. These were then mixed in different volume ratios to the same constant volume, usually 0.5 or 1.0 ml, to produce a set of reaction solutions run simultaneously in exchange experiments. Substrate levels were selected to be at or near the published K,,, values for each, except that [Pi] was somewhat higher, due to the highequilibriumconstantforthereaction at pH 7.8, 3O”C, (see legend, Fig. 1, Ref. 36). RESULTS

ModiJier eflects on equilibrium exchanges. In these experiments, substrate levels were held constant at or near their respective K, values, while modifier concentrations were varied. The effects of succinate, an inhibitor known to be directly competitive with aspartate (38, 39), were observed first, as 3 In this paper, the term “substrates” refers to all reacting species, e.g., for ATCase: Asp, C-Asp, C-P, and Pi. The term “reactants” refers to Asp and C-P, whereas “products” refers to C-Asp and Pi. The term “reaction components” includes all substrates plus salts, buffer, etc.

GASSER

shown in Fig. 1. The inhibition of native ATCase occurs such that the two exchange rates, R([32P]C-P s Pi) and R’([W]Asp % C-Asp) are suppressed essentially in eonstant ratio, i.e., (R/R’) is constant. Data of the accompanying paper (36) indicated an ordered binding mechanism for native enzyme. Comparing the inhibition pattern of Fig. 1 to model patterns (35) derived for OTdeTed systems, the modes of modifier action which alter R and R’ such that (R/R’) is essentially constant are: (a) where modifier competes directly for a substrate site, (b) where modifier blocks covalent interconversion, or (c) where modifier blocks association-dissociation, or dissociation only, of one substrate from each exchange being observed, e.g., Asp and Pi, or C-Asp and C-P. If one considers also the model inhibition patterns derived for random binding systems (35), those in which (R/R’) remains essentially constant are: (d) where modifier blocks covalent interconversion, steps, or (e) where modifier competes with one substrate from each exchange being observed. Cases (b)-(e) do not appear relevant, since the effect of succinate can be overcome by increased levels of aspartate alone and since succinate does not inhibit V under initial velocity conditions (38, 39). The mode of action most consistent with other data on the action of succinate is (a) for an ordered system. The inhibition also occurs in a cooperative manner (Hill n = 1.58), which strongly indicates direct binding at the n-Asp site. The effects of CTP are presented in Fig. 2. Over the range of O-O.4 mM concentration, CTP suppresses both R and R’ with slight cooperativity (Fig. 2B). The extrapolated values of l/i vs l/[M] in Fig. 2B are >l, indicating that incomplete inhibition of each exchange would result from modifier saturation in this mode of action. From earlier equilibrium binding studies (3, 38) these first sites are probably located on the regulatory subunits, but with [CTP] above 0.5 mM, it has been shown that direct competition with C-P and Pi at the active site region occurs (3). This mode of action leads to complete inhibition of both exchanges at infinite modifier levels, as seen by an intercept of 1.0 on the (l/i) axis at (l/[M]) = 0

MODIFIERS AND ATCase

o-

35 7 10

cr.5

71

25

IS

Succinate,mM

10 -

,,‘ , .*:,

7A, 4-

&’

I I

I I ’

I

i”+succinlte]

’

’

FIG. 1. Effect of varying succinate concentration on the kinetics of [32P]C-P % Pi (R, triangles) and (“C]Asp % C-Asp (R’, circles) exchanges at pH 7.8,3O”C, catalyzed by native aspartate transcarbamylase at equilibrium. (A) Rates, R and R’, vs [succinate] with (R/R’), (’Inset). Note scale differences. (B) Plot of (l/i) vs ]suceinate]-’ from A, using the equation (l/i) = KiC/‘[M] + V,/(V, - VI) as derived and discussed elsewhere (35). Substrate levels (in mM) were: 0.01 C-P, 5 Asp, 100 C-Asp, and 90 Pi. Enavme was used at 25 ~a ner ml of reaction volume. Isotopic labels were present at 0.05 &i [W]Asp, and 20 &i “Pi or 5 &i [32P]C-P. .Y_

in the reciprocal

plot of Fig. 2B. The ratio inset, Fig. 2A-decreases somewhat in the first portion of this biphasic inhibition process, then remains constant, as both R and R’ decrease. This behavior, compared to theoretical patterns for R, R’, and (R/R’), derived for compulsory order substrate binding systems (35), is not readily explained by single modes of modifier action. Modifiers which compete directly with or block association of substrates are shown to decrease R and R’ in constant ratio, but this is not observed for CTP < 0.4 InM. CTP could act, upon binding to the regulatory site, by inducing a protein conformational change which alters ~-Asp binding and also the association-dissociation rates or the binding of C-P and Pi. The recent observations of Rosenbusch and Griffin (46), indi-

(R/R’)-see

cating an interrelationship in C-P and ~-Asp binding to the enzyme, make this a reasonable possibility. Where [CTP] 2 0.4 mM, (R/R’) remains nearly constant. In this concentration range CTP has been shown by equilibrium dialysis to bind to the phosphate subsite of the active site region (3), and so should compete directly with C-P and Pi. A constant (R/R’) where modifier competes with substrate sites is observed only in compulsory order systems (35). In random systems, (R/R’) is constant only if modifier blocks covalent interconversion steps, which is not a mode of action observed for CTP by initial velocity or other techniques (36, see also below). The effect on equilibrium exchange rates of increasing levels of ATP is seen in Fig. 3A. The C-P % Pi rate is unchanged, but Asp e

72

WEDLER

AND

GASSER

I

a’

0.2

IO

0.4

0.6

I

0.8

1.2

I.6

.

041 8 1 0 4

1.4

e-

6

s:

2

-

e

H-3

I I 0

I 2

I

1

I

4

IO

IO-$/&,

I2

I4

M”

of varying CTP concentration on the kinetics of [32P]C-P = P, (R, triangles) and and C-Asp (R’, circles) exchanges at pH 7.8, 3O”C, catalyzed by native aspartate transcarat equilibrium. (A) Rates, R and R’, vs [CTP] with (R/R’), (inset). (B) Plot of (l/i) vs See Fig. 1 for other experimental conditions and explanations.

FIG. 2. Effect [“CJAsp bamylase [CTP]-1.

z

C-Asp is stimulated some 3- to 4fold. Consequently, R/R’ decreases. Since substrate levels in this experiment were not fully saturating (except for Pi) it remained to be shown whether the effect was on V or on the K, of Asp. Previous initial velocity studies (38) suggestedthat ATP lowers the dissociation constant for aspartate and abolishes the homotropic cooperativity associatedwith Asp binding. Therefore, Asp and C-Asp levels were varied in the presence and absenceof 8 mM ATP, as shown in Fig. 3B, and the Asp G C-Asp exchange observed at equilibrium. ATP is seento alter the binding curves rather markedly. A double-reciprocai plot of these data, (l/R’) vs (l/Asp) (see inset) indicates that V for Asp G C-Asp, not the “Km,” is altered by ATP. Thus, it appears that ATP increasesthe rates of Asp

and C-Asp dissociation, and diminishes cooperativity as well. Modifier e$ects on initial velocity kinetics. From earlier studies (14-17, 36) rate limitation at 30°C appears to be identified strongIy with a conformational change accompanying aspartate binding. This hypothesis was tested further by observing the effect of added ATP and CTP on various portions of the nonlinear Arrhenius plot presented previously (36). Figure 4 indicates that rates above 15*C are shifted markedly by added modifiers, whereas those below 15°C are changed much less drastically. The “break” of the plot is shifted by ATP to 27-28”C, whereas CTP leaves it unchanged. Above 15”C, with ATP present, the Arrhenius slope remains constant but the absolute magnitude of k is enhanced. The

MODIFIERS

73

AN 1 ATCase

is quite large and AS* is large and positive. This suggests that very different sorts of reaction pathways are traversed at 35°C and

6

T,“C

I

i

40

JO

3.2

3.3

20

0

10

I I. 5

3.0

2

4.5

6.0

7.5

ATP, mM

0.5 0.4 0.3 -

0

[Asp]

3.4 3.5 lOTOK

30

6 ,mE

1A~p/dfA~p=

0.1f

FIG. 3. Effect of varying ATP concentration on the kinetics of [3zP]C-P ti Pi (R, triangles) and (W]Asp % C-Asp (R’, circles) exchanges at pH 7.8, 3O”C, catalyzed by native aspartate transcarbamylase at equilibrium. (A) Rates, R and R’, vs [ATP]

(note scale difference), with (R/R’), (inset). rate in the presence and absence of 8 mM ATP, as a function of Asp and C-Asp levels, with C-P and Pi levels as described in Fig. 1. Inset shows l/R’ vs l/[Asp] for these data. (B) ASP % C-Asp

step. With CTP present, the slope of the plot above 15°C is altered and consequently so is E, and AH+. Table I presents calculated AH*, AG*, and AS* terms at 37°C. This comparison shows that CTP raises AH* and makes As* more positive, whereas ATP lowers AG+: and raises l~,,~ without such large changes in AH*. One interpretation of these results is that energetically the modes of action for ATP and CTP appear to be very different with E. coli ATCase. At 5”C, the AH* term

3.7

FIG. 4. Effect of ATP (7.5 mM, diamonds) and CTP (1.5 mM, triangles) on catalytic turnover rate of native ATCase &moles H+/min/Fg enzyme) in the Arrhenius plot, In k vs l/T, between 0 and 40°C. Data in the absence of ATP (circles) are taken from Fig. 5 of the accompanying paper (36). C-P was at 4 mM, with [Asp] at 50 mM at 25-4O”C, at 25 mM at l&25%, and 15 mM at 0-10°C (see text for explanation). Enzyme levels varied, depending on the temperature, from l-40 pg per experiment. TABLE

transition state is achieved more readily, primarily through decreases in the As+ and AG* terms. ATP also increases, therefore, the rate of Asp association, the rate-limiting

3.6

ACTIVATION

PARAMETERS

I ChLCUL.4TED

FROM THE

DOT,\ OF FIG. 4"

-

e3 37 37 37 5

Perturbant

0 ATP CTP (0, ATP CTP)

ASS (e.u.) 6.3 6.9 11.4 22.1

5.7 6.3 10.8 20.6

-27.4 -23.6 -10.3 f21.3

-tAsj

AG$

8.5 7.3 3.2 -6.6

14.2 13.6 14.0 14.0

I

a Expressed as kilocalories per mole. The term “k," originally expressed as rmoles prod/min/rg enzyme, was converted to units of set-I, a turnover number, for the calculation of AGj, assuming an enzyme M, = 300,000 daltons and 1007, active protein.

WEDLER AND GASSER

74

ATP -‘.

IO .

0

1

5

IO

15

20 25 L-Asp.mM

50

350

I

16

FIG. 5. Initial velocity rates as a function of aspartate concentration at 5°C (top) and 35°C (bottom) catalyzed by native aspartate transcarbamylrtse, in the presence and absence of modifiers. [C-P] was initially at 4.0 mM. k is expressed in pmoles of protons produced per minute per rg enzyme protein. Enzyme levels varied from 4-20 pg per experiment over 355°C.

at 0°C in accord with other data (36). To achieve the transition state for catalysis at O”C, the enzyme appears to need to become disordered, relatively. Low temperature thus may put the active site into an overly contracted and ordered state, unfavorable for catalysis. Kinetic studies of Asp binding in the presence and absence of CTP or ATP at 5°C I5”C, and 35°C were carried out also. Typical results at 5°C and 35°C are shown in Fig. 5. Asp binding is generally tighter at low than at high temperatures, as expected for a predominantly ionic association process. ATP clearly both stimulates V and lowers the S0.s value. The Asp binding curves with 7.5 mM ATP added show inhibition at higher

Asp levels. This may result from competition by Asp for anionic ATP, Pi, or C-P sites, forming dead-end complexes (4> 37, 39), or from binding to a separate modifier site (cf. Ref. 36, footnote 3). In order to obtain valid S0.s and n values from Hill plots of these data, one must take into account both the productive and nonproductive Asp complexation by first plotting (l/v) vs (l/s) to estimate I’, then using this value in the Hill equation: log 21 v-v

= n log [fl + log S&S. (1)

Unless this was done, calculated n values were too high or difficult to estimate.

MODIFIERS

AND

Interestingly, data from such Hill plots indicate that ATP does not completely abolish cooperative aspartate binding, as suggested previously (38) but in all cases, at all temperatures, n > 1. Hill plots made from the data of Gerhart and Pardee (18) confirm this also. Cooperative Asp binding with bound ATP is readily apparent in the curves at 5°C in Fig. 5. CTP, on the other hand, increases n and &.s for Asp, as reported previously. In these studies, K, values for Asp binding to catalytic subunits (7~ = 1) were also obtained at 5°C. 15”C, and 35°C. Figure 6A presents calculated values of the Hill coefficient n for Asp binding at each of the three temperatures, with and 4

A 35.

‘“~~, ‘\

3

b Htll

n

-

2

b ‘.

0

I \ \ ‘Cl 5, ‘A’ ’ .\ ’ fo\---

=---&

‘9,

loo

-

S,-,5, M-’

--------o-

300

2oo

1 25

log 5

-

rr

B

0.5 2.0

-

75

ATCase

without modifiers present, plotted against reciprocal SO.s values, as affinity constants. In general, this plot might be analogous to a potential energy diagram with isotherms, and resembles similar plots suggested earlier by Edelstein (40). One sees that at 15°C n responds maximally to added modifiers, but at 5°C n shows a minimal response. At 15°C. n is maximal for native enzyme compared to other temperatures. Catalytic subunits, it should be pointed out, exhibit a higher So.5or lower affinity for Asp than that of native enzyme with bound ATP. This prevents calculation of various allosteric parameters, as Edelstein (40) was able to do with hemoglobin, and implies that catalytic subunits do not represent a pure R-form of the enzyme. From the van? Hoff plot of log affinity constant (S,&) for Asp vs l/T, in Fig. 6B, one sees that catalytic subunits and native enzyme with bound CTP exhibit basically monophasic plots, but native enzyme alone or with bound ATP shows nonlinear plots suggesting (1) that native enzyme is more flexible conformationally with regard to ~-Asp binding than when converted to catalytic subunits or when CTP is bound, and (2) that ATP may induce a structure even more conformationally favorable for binding, such that the detrimental effect of higher temperature on Asp binding is somewhat reversed. This is evidenced by plot 6B being concave upward with ATP bound, instead of downward as for native enzyme. DISCUSSION

1.5 3.1

3.3

3.5

I03PK

FIG. 6. Effect

of modifiers upon the Hill coefficient, n, and the affinity constant, ~90.5, at assayed by the initial various temperatures, velocity technique. (A) Plot of the Hill n value vs the reciprocal SO.s value for aspartate, 80.5 in M-I, at 5, 15, and 35”C, in the presence of 7.5 mM ATP (diamonds), no modifier (circles), or 1.5 mM CTP (triangles). The &‘,., values for catalytic subunits are also shown (crosses). (B) van’t Hoff plot of log reciprocal SO.s (&.s), for aspartate at a function of reciprocal temperature, with catalytic subunits, native enzyme and native enzyme plus modifiers-legend for symbols as in part A. Data are calculated from Hill plots (see text).

The results presented above are in agreement with two basic conclusions. First, the inhibitory patterns produced with equilibrium exchange kinetics conform to those derived and predicted for an ordered substrate binding system with native ATCase at 30°C. Second, the modifiers ATP and CTP appear to alter the enzyme via separate mechanisms, not simply by perturbation in opposite directions of the equilibrium for aspartate binding. In regard to the first conclusion, this result provides additional verification of the conclusion derived from exchange kinetics varying substrate levels, reported in the accompanying paper (36), as well as the

76

WEDLER

AND GASSER

results of Stark and coworkers (37, 41). These results conflict with conclusions reached by Heyde et al. (39) with catalytic subunits at 28°C and by Silverstein (42) with native enzyme at O”C, for reasons discussed previously (36). In regard to the modes of action of ATP and CTP modifiers, current evidence in the literature is not completely in harmony as to whether the activator and inhibitor act to perturb in opposite directions the equilibrium associated with aspartate binding: Xl *

xz

(2)

or act via truly separate mechanisms. Consider the following observations: (a) Aspartate increases, but both ATP and CTP decrease, the rate of tryptic digestion (6) and sodium dodecyl sulfate-induced dissociation (43) of the protein. (b) The fluorescence data of Wong (24) indicate that substrates ATP and CTP each alter fluorescence and fluorescence decay time, energy transfer, and polarization in distinctly different ways for both intrinsic (Trp) and extrinsic (bound aminonaphthol sulfonate dye) probe molecules. (c) Equilibrium dialysis experiments by Gray et al. (44) indicated that binding of the transition state analog, PALA, reduced the affinity of the enzyme for CTP by 2-fold, but has no effect on ATP binding. (d) Heyde et al. (45) have observed that catalytic subunits partially digested with trypsin when recombined with regulatory subunits were activated by ATP but not inhibited by CTP. These are in contrast to (e), a correlation between structures of and protein effects induced by a series of nucleotides, that led London and Schmidt (29) to propose “contracted” and “expanded” states for the enzyme, stabilized by CTP and ATP, respectively. (f) Relaxation spectra, observed by Hammes and coworkers, are consistent with more than two conformational states being of importance in allosteric regulation (15). This has led to the proposal (16, 17) that substrates and ATP stabilize an active conformation, X1 in Eq. (2), whereas CTP stabilizes the other, inactive conformation, X2. Other interpretations of these relaxation spectra are possible, however. Because the catalytic and regulatory conformational changes involve

both types of subunits and are transmitted through the protein over considerable distances, any single approach may only be able to probe these phenomena in a particular limited region of protein structure. Hence, the difficulty in interpreting and integrating the above observations. For the data of Fig. 6A, Hill n values plotted vs Asp affinity, compared along isotherms, the simplest explanation is a mechanism like that of Eq. (2) as proposed by Hammes (16, 17). Other observations argue that the actual process is more complex, especially the data of Figs. 4 and 6B and of Table I. ATP and CTP appear to alter the rate and tightness of aspartate binding by very different mechanisms: ATP exerts an effect on rate (AG* and AS*), also evident in Figs. 3 and 5, whereas CTP alters the enthalpy (AH*) of the process. As an alternative mechanism the following concepts are proposed: consider first all the different processes known to occur in relation to aspartate binding at the active site. (1) Bound CTP can reversibly push into the Asp binding site a protein segment Z1 that interferes with the binding of Asp, increasing its Hill n and So.6 values. This heterotropic allosteric inhibition is basically a modifier-induced intramolecular (partial) competitive inhibition. (2) A.sp, upon binding, can overcome the CTP-induced effect and can force 21 out of the site reversibly. Ordered binding (36) implies that the binding of C-P may aid the binding of Asp. By analogy to succinate (38), this effect is at least some 30-fold, presumably by a minimum number of interactions. The data of Rosenbusch and Griffin (46) indicate that partially filled C-P sites are potentiated for complete saturation by the binding of succinate and probably L-ASP. (3) The binding of Asp moves a peptide segment 22 such that binding is improved and this effect is transmitted to other 2~ segments in adjacent subunits in the native enzyme by homotropic interactions (18, 38). (4) The binding of Asp also moves a pepfor tide segment 23 into juxtaposition catalysis and brings the a-amino group of Asp and the carbamyl group of C-P together (14, 37, 41).

MODIFIERS

(5) Covalent interconversion occurs. Are any of these segments 21, Z2, and Z3 the same, and which of the processes (l)-(5) may be the same or be coupled directly? Z1 and Z, may not be the same, since the data of Kerbirious and Her& (26) indicate that in a mutated enzyme, completely lacking homotropically cooperative Asp binding, high CTP may strongly alter the K, of Asp without restoring any cooperativity. Zz and Z3 may be at least adjacent segments, (or steps (3), (4), and (5) may be coupled), since several lines of evidence (14, 37, 41) suggest that some of the binding energy from aspartate association is used to induce a productive active-site conformation for catalysis. In agreement with this, even at low temperature, where catalysis is definitively rate limiting, although Asp binding and catalysis are energetically and thus kinetically separated, the Hill n is > 1. In summary, CTP appears to compete with Asp binding by binding at a separate distant regulatory site and reversibly pushing segment Z1 into the site. ATP, on the other hand, appears to make the kinetics of steps (3) and (4) more rapid, perhaps by making the active site more flexible or pushing their conformations toward the catalytically active one. One might consider that the energy of ATP binding is used to supplement that required from Asp, to reach step (5), so that bound ATP decreases the Hill n and S0.6 for Asp. The data of Figs. 3 and 4 imply that bound ATP enhances the rates of both association and dissociation for Asp. In regard to the recently proposed channel-access theory of regulation (30), our data do not support such a mechanism. Since (C-P + Pi) << (ASP % C-Asp) at 3O”C, neither the rate of access to the active site nor covalent interconversion can be rate limiting. If channel size were a critical parameter at 30-37”C, its diameter wouId presumably be poised to open or to constrict in response to substrates and ATP, or to CTP, respectively. The ability of ATP to stimulate only the faster exchange, (Asp + C-Asp), which involves the largest substrate, C-Asp, at 30°C (Fig. 3A) does not fit this picture. To rationalize this apparent conflict, one must invoke special properties of the channels and

AND

ATCase

77

central cavity that would differentially retard the flux of C-P and Pi. Since CTP alters only the So,, and Hill n of Asp, not V, the channel access hypothesis seems even more in doubt. Jacobson and Stark (47) have shown that “native” enzyme deficient in regulatory subunits4 still has sensitivity to CTP. At low temperature, if channe1 size does control net turnover rate, modifiers do not perturb this process in a manner markedly different from that observed at 35°C (cf. Figs. 4 and 5). For example, one might expect, but does not observe, superactivation by ATP at 5°C. Once detailed crystallographic pictures for nat.ive enzyme n-ith and n-ithout bound substrates and modifiers are available, it will be perhaps more evident whether or not the above proposals represent realistic working hypotheses. ACKNOWLEDGMENTS The authors thank Drs. Todd Schuster and W. M. Lipscomb for stimulating discussions, and Mrs. JoAnn Cencula for her excellent technical assistance. We also acknowledge support for this research from the National Science Foundation, Grant GB 34751. REFERENCES 1. JACOBSON, G. It., AND STARK, G. R. (1973)in The Enzymes (Boyer, P. I~., ed.), 3rd ed., vol. 9, pp. 225-308, Academic Press, New York. 2. COOK, R. A. (1972) Biochemistry 11, 3792-3797. 3. MATsUMOTO, S., AND HAMMES, G. G. (1972) Biochemistry 12, 1388-1394. 4. COLLINS, K. D., AND STARK, G. R. (1969) J. Biol. Cherrt. 244, 186991877. 5. HAMMES, G. G., PORTER, R. W., AND WV, C.-W. (1970) Biochemistry 9, 2992-2994. 6. MCCLIXTOCK, D. K., AND MARCUS, Q. (1968) J. Biol. Chem. 243.2855-2862. 7. MCCLINTOCK, D. K., AND MARCUS, G. (1969) J. Biol. Chem. 244, 36-42. (1972) J.Biol. 8. COLMAN, P.D., ANDMARCUS,G. Chem. 24’7, 3829-3837. Q C. KIRSHEER, M. W., AND SCHACHMAN, H. K. (1971) Biochemistly 10, 1919-1926. 10. COHLBERG, J. A., PIGIET, V. P., JR., AND SCHACHMAN, H. K. (1972) Biochemistry 11, 3396-3411. 11. VANAMAN, T. C., AND STARK, G. R. (1970) J. Biol. Chem. 246, 3565-3573. 4 This species has been characterized (G. It. Stark, personal communication).

as CeRr

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