Aspartate Transcarbamylase · to study the catalytic subunit first, in order to avoid the ... In this paper, we discuss the steady state kinetics of the interactions of the catalytic

THE JOURNAL OFBIOLOGKXL CHEMISTRY Vol. 244, No. 7, Issue of April 10, pp. 1846-1859, 1969

Printed in U.S.A.

Aspartate Transcarbamylase

KINETIC STUDIES OF THE CATALYTIC SUBUNIT*

(Received for publication, September 26, 1968)

ROBERT W. PORTER, MICHAEL 0. MODEBE, AND GEORGE R. STARK

From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 9@05

SUMMARY

1. Steady state kinetics indicates that the binding of substrates by the catalytic subunit of aspartate transcarbamylase is ordered. Product inhibition patterns show that carbamyl phosphate binds first, aspartate binds second, carbamylaspartate dissociates first, and phosphate dissociates second.

2. Nonlinear product inhibition and substrate inhibition indicate that several dead end complexes are formed: aspartate binds to the enzyme-phosphate complex, a 2nd mole of phosphate binds to the enzyme-phosphate complex, a 2nd mole of carbamyl phosphate binds to the enzyme-carbamyl phosphate complex, and carbamylaspartate binds weakly to the free enzyme.

3. The interaction of the catalytic subunit with analogues of both substrates has been studied in an attempt to reveal those structural features of the substrates that are required for binding and function. Acetyl phosphate and N-methylcarbamyl phosphate are the only analogues of carbamyl phosphate tested which are substrates, with maximum veloci- ties at pH 7.8 of 2.4% and 0.03% of the maximum velocity with carbamyl phosphate. Since acetyl phosphate lacks the NH2 group of carbamyl phosphate, this group cannot be essential for function. N, N-Dimethylcarbamyl phosphate is not a substrate, but it is a competitive inhibitor, as is any analogue of carbamyl phosphate with a phosphate or phosphonate dianion, suggesting that at least part of the site for carbamyl phosphate is readily accessible. In fact, with the catalytic subunit, even cytidine triphosphate is a competitive inhibitor.

4. Several dicarboxylic acids were found to be competitive inhibitors of L-aspartate. Succinate and maleate are the strongest inhibitors, malonate and D- and L-malate are good inhibitors, whereas fumarate, glutarate, and D- and L-bromosuccinate are poor inhibitors, and D-aspartate does not inhibit significantly.

5. The pH dependence of the dissociation constant for succinate indicates that a group with pK, 7.1 is required to

* This investigation was supported by Grant GB 7813 from the National Science Foundation and by Public Health Service Research Grant GM 11788 from the National Institute of General Medical Sciences.

be positively charged for this inhibitor to bind to the enzyme carbamyl phosphate complex. In contrast, the dissociation constants for carbamyl phosphate and phosphonacetamide vary little between pH 6 and pH 9.

6. Product inhibition by carbamylaspartate when aspartate is varied indicates that the constant for dissociation of aspartate from the central complex is much larger than the dissociation constants for unreactive aspartate analogues such as succinate. This indicates that some of the binding energy of aspartate may be used to facilitate the reaction with carbamyl phosphate.

7. The catalytic subunit is unstable at concentrations below 1 pg per ml. There is an immediate decrease in specific activity with decreasing enzyme concentration and a slower irreversible inactivation during incubation at low concentrations. Both the immediate and the slow losses of activity are prevented by the presence of bovine serum albumin (50 pg per ml) in dilute enzyme solutions. However, because bovine serum albumin forms a complex with the catalytic subunit and alters the apparent kinetic parameters, it was not used in these kinetic studies.

8. At pH 7.8 and 28”, the dissociation constant for carbamyl phosphate is 1.4 X 1OF M, the K, for aspartate is 0.020 M, and the V,, is 9.0 X lo4 moles per min per 100,000 g of subunit. The dissociation constant for carbamyl phosphate and K, for aspartate vary little between pH 6.4 and pH 8.7, whereas Vm, changes greatly, with an optimum near pH 8. With the use of acetyl phosphate instead of carbamyl phosphate, K, for aspartate is unchanged at pH 7.8, but the dissociation constant for acetyl phosphate is 35-fold higher. With N-methylcarbamyl phosphate, G for aspartate is 7-fold higher and the dissociation constant for N-methylcarbamyl phosphate is 4-fold higher than for carbamyl phosphate.

The aspartate transcarbamylase of Escherichia coli catalyzes the condensation of L-aspartate and carbamyl phosphate to form carbamyl-L-aspartate, the first product along the pathway of pyrimidine biosynthesis that leads eventually to cytidine triphosphate. The enzyme was one of the first shown to be subject to

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metabolic control by feedback inhibition: it is inhibited specifi- cally by CTP (1). Catalytic activity and affinity for CTP reside in different subunits, separable after treatment with p-hydroxymercuribenzoate (2).

This paper and the two accompanying it (3, 4) are the initial reports in an attempt to define the mechanism of action of aspartate transcarbamylase in detail. An understanding of the catalytic mechanism is not only intrinsically interesting, but also is an essential part of an understanding of the mechanism of feedback inhibition for this enzyme. We have decided to study the catalytic subunit first, in order to avoid the kinetic complications of homotropic interactions of the native enzyme with aspartate (1) and to simplify the system chemi- cally. Our conviction that the catalytic mechanisms of the subunit and the native enzyme are essentially ident’ical is but- tressed by the similarity of limiting kinetic constants for the two forms (1). In this paper, we discuss the steady state kinetics of the interactions of the catalytic subunit with carbamyl-Pl and aspartate and with analogues of the normal substrates. The accompanying papers include studies of enzyme-substrate and enzyme-inhibitor interactions by nuclear magnetic resonance (3) and by ultraviolet difference spectroscopy (4), and a working hypothesis for the mechanism of action (4).

MATERIALS AND METHODS

Enzyme-The catalytic subunit of aspartate transcarbamylase was prepared according to Gerhart and Holoubek (5). For kinetic studies the enzyme was removed from phosphate buffer by dialysis into either 0.05 M imidazole acetate, pH 7, or 0.05 M

sodium glycylglycine, pH 8, containing 2 X lop3 M 2-mercapto- ethanol and 2 X 1OV M EDTA. Different preparations of enzyme had very similar properties.

Carbamyl-P and Its Analogues-Carbamyl-P monohydrate (Sigma) was used without purification. The purity reported by the manufacturer (greater than 90%) was confirmed by analysis for labile phosphate (6). Sodium phosphate and sodium pyrophosphate were commercial reagent grade chemicals. CTP was obtained from P-L Biochemicals.

Acetyl phosphate was synthesized in a new way so that it would be free of inorganic phosphate. An excess of redistilled acetic anhydride (0.1 mole) was reacted with the monotriethylammonium salt of anhydrous phosphoric acid (0.05 mole) in an anhydrous acetonitrile solution (50 ml). After 1 hour at room temperature, acetonitrile, acetic acid, and unreacted acetic anhydride were removed with a rotary evaporator, and the oily residue was dissolved in 50 ml of methanol. The dilithium salt of acetyl phosphate was precipitated by adding 50 ml of methanol containing 0.11 mole of lithium acetate. The product was assayed by complete enzymatic conversion with excess aspartate transcarbamylase to N-acetyl-14C-L-aspartate and for total phosphate after acid hydrolysis (7). These tests indicated that the product was at least 90% pure and free of appreciable inorganic phosphate.

Phosphonacetic acid was prepared from the triethyl ester (Aldrich) by hydrolysis in 6 M HCl for 3 hours at reflux. Phos- phonacetamide was prepared according to Balsiger, Jones, and Montgomery (8) and both compounds were recrystallized until

1 The abbreviations used are: carbamyl-P, carbamyl phosphate; BSA, bovine serum albumin.

their melting points agreed with the values reported by these authors. In the synthesis of phosphonacetamides from phosphonacetyl chloride and amines, a yeIlow impurity was always present. We removed it by passing the impure product through a column, 2 x 50 cm, of Dowex 50-X8 200 to 400 mesh, hydrogen form in water. With water as eluent, the yellow impurity pre- ceded the derivatives by about 20 ml. N-Methylphosphonacet- amide, prepared from methylamine and phosphonacetyl chloride, was purified in this way and then recrystallized from glacial acetic acid to a melting point of 121-123”. The dipotassium salt of N-(p-nitrophenyl)phosphonacetamide, prepared by adding 2 eq of p-nitroaniline to 1 of phosphonacetyl chloride, was recrystallized from water after chromatography as described above, but with the potassium form of Dowex 50 in water. The yellow product had a maximum absorbance at) 319 m,u a.nd EJ~ = 15,000. Dilithium methylphosphonate was prepared from the commer- cially available dimethyl ester (Aldrich) by hydrolysis in 6 M

HCl. After the acid had been removed with a rotary evaporator, the oily product was dissolved in water and neutralized to the phenolphthalein end point with saturat’ed LiOH. Addition of an equal volume of ethanol caused precipit’ation of the dilithium salt.

Monomethyl phosphate and monoethyl phosphate were synthesized with trichloroacetonitrile by the method of Cramer and Weimann (9), except that anhydrous methanol or ethanol was used as the solvent in place of acetonitrile. The monoesters were purified on a column, 2 x 50 cm, of Dowex 50, hydrogen form in water. One drop of phenolphthalein was added to each 5-ml fraction from the column, followed by saturated LiOH, dropwise, to the end point. Tubes containing appreciable amounts of acidic material were pooled, evaporated to dryness, and redissolved in a minimum volume of water. Dropwise addition of an equal volume of ethanol caused precipitation of the dilithium salts. After drying under reduced pressure over PzOh, the products gave the expected neutralization equivalents, contained less than 0.2% of free phosphate, and liberated 95 to 100% of the expected amount of phosphate after hydrolysis overnight in 6 M HCl at 110”.

N-Monosubstituted carbamyl phosphates were synthesized according to the general method of Cramer and Winter (lo), in which anhydrous triethylammonium phosphate in acetonitrile is mixed with 1 eq of the appropriate isocyanate, whereupon the product precipitates. N-Phenyl-, N-(p-methoxyphenyl)-, N- (a+naphthyl)-, and N-(p-nitrophenyl)carbamyl phosphates were prepared successfully by this procedure, but no precipitate was obtained with p-tolyl- and o-methoxyisocyanates. All of the derivatives were rather labile; the dry solids decomposed within several months when stored at room temperature. Because of lability and the poor solubility in water of these aromatic compounds, only the p-nitropheny1 derivative was used in investi- gations with the enzyme. At 25”, N-(p-nitrophenyl)carbamyl phosphate, monotriethylammonium salt, has a half-life of 2.5 hours. The half-life is only about 0.5 hour at pH 8 and 28”, the conditions used for determination of K; values. Decomposition was followed spectrophotometrically: the carbamyl phosphate has a maximum absorbance at 323 nip, EM = 12,300, whereas p- nitroaniline, the hydrolysis product, has its maximum at 380 rnp and a similar extinction coefficient,

N-Methylcarbamyl phosphate was prepared as the dilithium salt, as described by Allen, Richelson, and Jones (11). How- ever, the crude reaction product contained so much dilithium


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1848 Kinetic Studies of Aspartate Transcarbamylase Vol. 244, No. 7

phosphate that it could not be purified by recrystallization from cold ethanol. To remove the excess phosphate, a portion of the reaction product was chromatographed at 4” on a column, 2 x 20 cm, of Dowex 2-X8, formate form, with a gradient of ammonium formate, pH 5.0, from 0 to 0.5 M with a l-liter mixing ves- sel. A peak containing heat-labile organic phosphate followed the peak containing free phosphate. Ammonium ion was ex- changed for lithium on Dowex 50, and the dilithium salt was precipitated by the addition of ethanol. Analyses for free and labile phosphate, quantitative determination (on the amino acid analyzer) for methylamine released by acid hydrolysis, and complete enzymatic conversion to N-methylcarbamylaspartate all indicated that the product was 90% pure.

Balsiger et al. (8) prepared N,N-dimethylcarbamyl phosphate as the magnesium salt but, because their product was very labile, they identified it by infrared spectroscopy only. We have prepared the dipotassium salt, which is much more stable; the half- life at pH 8 and 60” is 60 min. Our synthesis combines features of Balsiger’s and Cramer’s (10) methods: triethylamine and anhydrous HaPOd, 0.05 mole each, were dissolved in 40 ml of acetonitrile. To the homogeneous solution was added 0.053 mole of dimethylcarbamyl chloride (Eastman) and the mixture was re- fluxed for 1 hour. Triethylamine (0.1 mole) and 20 ml more of acetonitrile were added and refluxing was continued for 30 min more. Upon cooling to room temperature and concentrating to approximately 0.5 volume, white crystals of triethylammonium chloride separated and were removed by filtration. Fur- ther evaporation of the filtrate yielded an oil. One-half of the oil was dissolved in 10 ml of water, neutralized with KOH to the phenolphthalein end point, and chromatographed on Dowex 50-X8, K+ form in water at 4”. The combined peak tubes contained 0.23 M labile phosphate and 0.04 M free phosphate. The solution was divided into small portions and stored at -20”.

Analogues of Asp&&e-Commercial preparations of succinate, maleic, fumaric, malonic, glutaric, n-malic, and D-IdiC acids were recrystallized before use. Commercial crystalline D-&S-

partate was not purified further. D- and n-Bromosuccinic acids were synthesized by Dr. Guy Her& according to the procedure of Holmberg (12). The values of [ar], for DL pairs of optically active compounds were equal and opposite at pH 7.0.

Carbamyl-n-aspartate was prepared by enzymatic synthesis. Catalytic subunit (0.03 mg per ml) was added to carbamyl-P (0.1 M) and n-aspartate (0.125 M) in 30 ml of 0.2 M Tris acetate, pH 8.5. After 1 hour at room temperature, the volume of the reaction mixture was reduced by evaporation in a vacuum; the resulting concentrate was applied to a column, 2 x 50 cm, of Dowex 50-X8, hydrogen form in 1.5 M acetic acid at 4”. Fifty 6-ml fractions were collected and assayed for phosphate (7) and carbamylaspartate (13). A peak of phosphate was followed by a well separated peak of carbamyl-n-aspartate. The fractions containing carbamylaspartate were pooled and lyophilized. The amount of carbamylaspartate determined calorimetrically agreed with the amount of aspartate released upon alkaline hydrolysis (14).

Determination of Enzymatic Activity-The calorimetric assay used by Gerhart and Pardee (1) is not well suited to careful kinetic analysis. A more sensitive, more accurate, and more general method was developed, based on the use of 14C-labeled n-aspartate and the isolation of 14C-labeled carbamylaspartate from small columns of Dowex 50. Unlike the calorimetric assay,

this method is sensitive to any reaction in which the amino group of aspartate is acylated, and was used with N-methylcarbamyl phosphate and acetyl phosphate as well as with carbamyl-P. Although our procedure was developed independently, it is very similar to the one already described by Bresnick and Moss6 05).

The reaction mixture (0.5 ml) contains carbamyl-P or an analogue, l*C-aspartate (adjusted to the appropriate pH), enzyme diluted with the same buffer used for dialysis, and buffer. The buffer was usually 0.2 M Tris acetate, imidaeole acetate, or sodium glycylglycine; sodium cacodylate was also used occasionally. None of these are inhibitory at 0.2 M. (Kleppe (16) has observed that chloride and several other common anions inhibit the enzyme at high concentrations and should be avoided.) At appropriate times, 100 &portions of the reaction mixture are with- drawn and pipetted into 1.5 ml of 0.2 M acetic acid that has previously been placed atop a small (0.4 x 5 cm) column of Dowex 50-X8, 200 to 400 mesh, H+ form in water. (The H+ form of the resin is prepared batchwise by washing with NaOH, HCI, and water. A 2 M HCI wash of a small portion of resin prepared in this way should contain no appreciable salt.) Acetic acid is started through the column by air pressure and then allowed to flow by gravity. Three rinses of water, 1.0 ml each, are then placed on each column and allowed to flow through by gravity or blown through with air pressure. The effluent is collected directly in vials and evaporated to dryness at 80”. The residue in the vial is dissolved in 1 ml of water; then 20 ml of scintillation fluid (17) are added. The samples are counted (efficiency of about 65%) along with a portion of the reaction mixture that has not been passed through a column of Dowex 50.

L-Aspartic acid, uniformly labeled with i4C, specific activity about 150 mCi per mmole (New England Nuclear), is diluted to the desired specific radioactivity with unlabeled n-aspartate. In order to remove a small amount of radioactive impurity and thus improve the background, the 14C-aspartate is adsorbed to a column, 0.9 x 5 cm, of Dowex 50-X8, H+ form in water, and, after washing the column with water, eluted with 2 M HCI and recovered after evaporation of the solvent. Labeled aspartate can be recovered as follows. The resin from a large number of experiments is combined and washed on a Buchner funnel with water. The aspartate is eluted with several portions of 2 M HCI. (If an appreciable amount of NaCI, from incomplete preliminary washing of the Dowex 50 or from buffers that contain sodium ions, is present in this wash, the aspartate should be separated from the bulk of the salt before the next step by triturating the dry residue with small portions of 12 M HCl until no appreciable radioactivity remains in the solid phase.) After removal of solvent, the residue is dissolved in 20 ml of water and applied to a column, 2 x 23 cm, of Dowex l-X8, 200 to 400 mesh, acetate form in water. Elution with 150 ml of 1 M acetic acid, followed by 1.5 M acetic acid, gives a small peak of radioactivity at approximately 60 effluent ml (discarded) and a large peak of 14C-aspartate at about 225 ml. Evaporation of the latter to dryness gives a white crystalline product, free of buffer salts.

Calculations and Data Processing-Initial rates were calculated by a least squares linear fit of the experimental data, performed by digital computer. An IBM 360/50 time-sharing system with on-line communication was used, and programs were written in the PL/ACME language, a variation of PL/l. Initial rates, based on four time points, were linear when up to 10% of the


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limiting substrate was consumed. Standard deviation from a I I I straight line was usually less than 2%. The specific radioactivity of 14C-n-aspartate was selected to give a minimum of 500 cpm l

for conversion of 10% of the limiting substrate. 0.4 - 2 l 0

Kinetic data were analyzed in two ways (18). First, recipro- ---------------------.-----------.

cal plots of initial velocity against concentration of the variable t- 0 0

0 substrate (1 /V versus 1 /S) were made and inspected for linearity and form. Second, the data were treated by a curve-fitting program for an appropriate hyperbolic function (V versus S), indicated by the form of the reciprocal plot, using as initial estimates,

E e

values of the appropriate parameters that were calculated 9 from the reciprocal plot. For example, for a single variable substrate, the initial rate follows the simple Michaelis-Menten function, Equation 1:

;:$---

s v = vmnx - Km + S

The program uses trial and error to fit data to this simple hyperbolic function with two parameters and makes special use of the on-line communication available with the ACME system. The reiterative procedure used completes the calculation of the parameters for optimal fit, to an accuracy exceeding that of the data, in less than 10 set of computer time in a time-sharing en- vironment. Similarly, more complex hyperbolic functions with more parameters, such as the equations describing inhibition

3.0 Enzyt2 concentra+io~(/ug/tnl)

patterns, are used for curve-fitting where indicated, with corres- pondingly longer times for calculation.

0.01 & carbamyl-P and ‘0101 M asparate. 0; &bout BSA; 0, with BSA, 50 pg per ml.

FIG. 2. Specific activity plotted against concentration of catalvtic subunit. Assav. 0.2 M Tris acetate. uH 8. at 28” with

I I I i 2 4 8

Hours & preincubation

RESULTS AND DISCUSSION

Some Properties of Enzyme at High Dilution-Initial experiments with the catalytic subunit showed first that the enzyme is slowly and irreversibly inactivated in dilute solutions (Fig. l), and second that, below a concentration of 1 pg per ml, the specific activity decreases immediately with decreasing enzyme concentration (Fig. 2). The decrease in activity at low enzyme concentration has also been observed by Bethel1 et al. (19). Per- forming the assay in plastic rather than glass test tubes prevented neither the immediate decrease in specific activity nor the slower inactivation. No significant activity remained after 24 hours of preliminary incubation when several conditions different from those of Fig. 1 were tested. For example, preliminary incuba- tions were done at 4’ rather than 28”, at 1.2 rather than 0.4 pg of enzyme per ml, with sterilized solutions at 28” and 4”, or with 0.04 M phosphate buffer in place of Tris acetate. (Phosphate has been shown to stabilize the enzyme at higher concentrations (5).) However, bovine serum albumin completely abolished inactivation and actually increased the specific activity at all concentrations of catalytic subunit (Fig. 2). The protective effect is dependent on the concentration of BSA: at either 0.4 pg per ml or 2.0 E.cg per ml of catalytic subunit, 70% of the original activity remained after 24 hours of preliminary incubation at 28” with 10 pg per ml of BSA and 100% remained with 30 pg per ml

FIQ. 1. Rate of inactivation of aspartate transcarbamylase of BSA. The enhancement of activity in the presence of BSA catalytic subunit in dilute solution. Percentage activity remaining plotted against time of preliminary incubation. Preliminary

(Fig. 2) involves changes in both K, and V,,,. With 2 pg per

incubation, catalytic subunit (0.4 pg per ml) in 0.2 M Tris acetate, ml of enzyme, the iY, for aspartate was lower and the limiting pH 8, at 28”. Assay, catalytic subunit (0.2pg per ml) in 0.2 M Tris J’max was higher in the presence of 50 pg per ml of BSA. BSA acetate, pH 8, at 28”, with 0.01 M carbamyl-P and 0.01 M aspartate. was not used in the kinetic studies reported below because of


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1850 Kinetic Xtudies of Aspartate Tyanscarbamylase Vol. 244, No. 7

these indications that it forms a complex with the catalytic subunit.2

Determination of Kinetic Parameters-Trial experiments showed the expected convergence of double reciprocal plots but also revealed a problem: K, for carbamyl-P is about low5 M,

while K, for aspartate is much higher (0.02 M). This situation makes the usual technique of varying the concentrations of both substrates in the ranges of their respective K, values very dilh- cult experimentally, since the assay with 14C-aspartate would require separating a very small amount of radioactive product (lo-” M or less) from a much larger amount of unused 14C-aspartate (0.02 M). The assay also requires the use of high concentrations of 14C-aspartate of very high specific radioactivity (5 x lOI cpm per mole) in order to provide at least 500 cpm for low6 M product in a O.l-ml aliquot of the reaction mixture. Furthermore, the initial rates at low carbamyl-P and high aspartate concentrations were so fast that more than 10% of the carbamyl-P was consumed before four aliquots could be taken at lo-see intervals, unless a very low concentration of enzyme was used. However, as noted before, the catalytic subunit is unstable at concentrations below 1 pg per ml, and its specific activity varies with enzyme concentration.

Therefore, a new scheme for determining kinetic parameters was devised. Equation 2 shows the expected dependence of initial rate upon concentrations of the two substrates for a sequential reaction mechanism.3

AB v = vnmx KiJh + AKb + BKo + AB

(2)

Equation 2 may be rearranged to the form of Equation 2a:

A v = v,, - *

B K, + A A + Kia

& . . I_ + B CM A + KG

I f the concentration of Substrate B is varied and Substrate A is held constant, and the resulting initial rate data are fitted to the

2 The concentration dependence of the immediate decrease in specific activity and the time dependence of the slower inactivation suggest the following hypothetical model for these two related effects.

dissociated slow denatured Catalytic subunit + polypeptide -, chains chains

Catalytic’subunit:BSA

According to this model, the dissociated chains are inactive, and the specific activity is determined by the concentration of intact catalytic subunit. The dissociated chains can undergo slow and irreversible denaturation. The over-all rate of inactivation will depend on the concentration of dissociated chains, which increases with increasing total enzyme concentration (but decreases in proportion to the concentration of intact catalytic subunit). I f the irreversible denaturation is slow, the rate of inactivation will also depend on the average lifetime of a dissociated chain, which decreases with increasing enzyme concentration. Forma- tion of a stable complex with BSA would shift the equilibrium away from the dissociated state.

3 The terminology used is that of Cleland (20): sequential, both substrates add to the enzyme before either product is released; ordered BiBi, two substrates add in obligatory order before any products are released and products also leave in obligatory order; ping pong, one or more products are released before all substrates have added to the enzyme.

simple hyperbolic function of Equation 1, the following apparent kinetic parameters are obtained:

A apparent V,,, = V,,, -

Ka + A

A +&a apparent Km = Kb ~ A + Ka

If the constant concentration of Substrate A is saturating, then Equation 3a indicates that the apparent V,,, is in fact an excellent approximation of the limiting I’,,,, and Equation 3b indicates that the apparent K, is an excellent approximation of Kb. Because an excess of carbamyl-P is used in this experimental system, high concentrations of product and high levels of product counts may be obtained, even when r4C-aspartate of low specific radioactivity is used (loll cpm per mole).

Alternatively, Equation 2 may be rearranged to the form of Equation 2b :

B v = Tr,, -

A & + B K, & + BKaIKia

+A G’b)

za Kb + B

Now, if the concentration of Substrate A is varied while that of Substrate B is held constant, and the rate data are fitt.ed to Equa- tion 1, the following apparent kinetic parameters are obtained:

B apparent V,,, = V,,, -

Kb + B

apparent Km = Kg, Kb + BKJK, Kb + B

(4b)

If the constant concentration of Substrate B (aspartate) is very low (10e5 M), the rates are sufficiently slow so that the concentration of carbamyl-P may be varied in the range of 10e5 M when the enzyme concentration is 2 pg per ml without the consump- tion of more than 10% of either substrate. With this scheme, a greater fraction of the aspartate can be converted to product. Equation 4a indicates that, under these conditions, the apparent vm,, obtained by fitting the data to the simple function of Equa- tion 1 is in fact V,, (B/Kb), and Equation 4b indicates that the apparent K, is an excellent approximation of Ki,.

The experiments to determine V,,, Ki,, and Kb were conducted at 28” and at four different pH values: pH 6.4 (0.2 M imidazole acetate), pH 7.0 (0.2 M imidazole acetate), pH 7.8 (0.2 M sodium glycylglycine), and pH 8.7 (0.2 M sodium glycylglycine). The data from one of these experiments are plotted in Fig. 3, and the results from all four are summarized in Table I. These results indicate that Ki, and Kb vary only slightly between pH 6.4 and pH 8.7, but that ‘CT,,, increases sharply between pH 6.4 andpH 7.8, withan optimum near pH 8. Since the reactive form of aspartate is undoubtedly the one with the amino group un- charged, the pH dependence of V,,, reflects both the pK, of this amino group and the pK, values of enzyme side chains or of enzyme-substrate intermediates in the rate-determining step.

Tests for Cooperativity-With the very sensitive assay for aspartate transcarbamylase it was confirmed that the catalytic subunit exhibits simple Michaelis-Menten kinetics at low substrate concentrations. The data shown in Fig. 3 show the excellent fit of the rate data to the hyperbolic function of Equation 1 when carbamyl-P is varied at limiting aspartate. A similar hyperbolic


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0.6

0.6

r- linearly with aspartate concentration, and that the slope of this linear portion equals apparent Vmax/&,, as expected for the hy-

h perbolic function of Equation 2a, where the apparent V,,, is the limiting V,,, for saturating carbamyl-P and is V,,,/2 for half-saturating carbamy-P (Equation 3a). Furthermore, this initial slope extrapolates perfectly into the hyperbola for higher aspartate concentrations, as shown in the inset for Fig. 4.

I 50

I I 100 150

CAP (PM)

I I 200 25

Product Inhibition-Using equilibrium dialysis, Changeux, Gerhart, and Schachman (21) have found that succinate, an unreactive analogue of aspartate, binds tightly to aspartate transcarbamylase only in the presence of carbamyl-P. They have therefore suggested an ordered mechanism for aspartate transcarbamylase. As Cleland (20) has pointed out, an ordered BiBi mechanism can be distinguished from other possible mechanisms by a thorough study of product inhibition, in which each substrate-product pair is varied while the other substrate is either limiting or saturating. The pattern of inhibition types obtained is unique for a particular mechanism. Product inhibition experiments were conducted at 28” and pH 7.8 in 0.2 M sodium glycylglycine. The results are shown as reciprocal plots in Figs. 5 to 8. For reasons discussed above, a rate-limiting concentration of aspartate was used for those experiments in which carbamyl-P concentration was varied through the range of its apparent K, (Figs. 5 and 6), and excess carbamyl-P was used when aspartate was varied through a similar critical range (Figs. 7 and 8). In the experiment shown in Fig. 7, the concentration of Substrate R (carbamyl-P), which would be saturating in the absence of Product Q (phosphate), is not saturating at the high

FIG. 3. Initial rate plotted against concentration of carbamyl-P concentrations of Q used. (CAP) in 0.2 M sodium glycylglycine, pH 8.7, at 28”, with 2 X lo+ M aspartate and 2 fig per ml of catalytic subunit.

An ordered BiBi mechanism predicts noncompetitive inhibition for all of the combinations tested except the one in which

TABLE I I I I I I

Kinetic parameters for catalytic subunit 1.4

Assay conditions were 28’ and 0.2 M buffer: imidazole acetate at pH 6.4 and 7.0, sodium glycylglycine at pH 7.8 and 8.7. Con- centration of catalytic subunit was 2 pg per ml. Values of vm,,, 1.2 Kb, and Ki were determined at saturating carbamyl-P (0.01 M).

Values for Ki, were determined at rate-limiting aspartate concentrations: 5 X 10e5 M at pH 6.4, 2 X 10e6 M at pH 7.0, 1 X 10e5 “0 M at pH 7.8, and 2 X 1W6 M at pH 8.7. - 1.0 .,

80

PH &a Kb

"0 60

x 40 >

20

0

I.r‘+f 16 27 14 37

PlZM

11 20 20 20

rt5

f $0.8

6.4 7.0 7.8 a.7

Estimated error (%I

f10

0.46 0.36 0.17 0.10

0.087 2 0.47 E 0.90 0.61

-g 0.6

E

f5 > 0.4 f20

a Kc is the apparent inhibition constant for substrate inhibition by aspartate, according to Equation 6. (See text for further details.)

curve has been observed by Bethel1 et aZ. (19). To detect possible cooperativity of the catalytic subunit for aspartate, aspartate

bU

concentration was varied through a very low range, first with saturating carbamyl-P (lop3 M) and then with half-saturating FIG. 4. Initial rate plotted against concentration of aspartate

carbamyl-P (2.5 X 1O-5 M) (Fig. 4). The data shown in Fig. 4 in 0.2 M imidazole acetate, pH 7.0, at 28”, with 2 pg per ml of cata-

clearly indicate that, at aspartate far below Kb, the rate increases lytic subunit. 0, 1 X 10-a M carbamyl-P (saturating); 0, 2.5 X low6 M carbamyl-P (half-saturating).


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1852

1200

Kinetic Studies of Aspartate Transcarbamyhse Vol. 244, No. 7

I Algebraic derivation shows that, for the ordered BiBi mechanism represented in Fig. 9, the displacement of the intersection from the ordinate equals

b ks + kc -.- k4 ke

where k) and k4 are the individual rate constants for binding and dissociation of Substrate B (aspartate), and kr, and ke are the rate constants for the chemical step in the forward and reverse direc- tions. If, as observed, this displacement is too small to be de- tected, then the on rate constant (ka) must be small compared to the off rate constant (k4).

25

O 0 I I I

100 200 400 20 i/CAP (,uM)-’ ‘:

FIG. 5. Product inhibition by carbamylaspartate, varying -z carbamyl-P (CAP) at constant aspartate (2 X 10-d M). l/V e I5 versus l/carbamyl-P. Carbamylaspartate concentrations: 0, $! 0.0207 M, 0.0414 M, and 0.0621 M. Sodium gIycyIgIycine, 0.2 M, pH 7.8, at 28”, with 2pg per ml of catalytic subunit.

g $0

I I I I I I I 2

Y-4

0 0 50 100 150 200 250 l/Asp (M)-’

FIG. 7. Product inhibition by phosphate, varying aspartate at constant carbamyl-P (0.01 M). l/V versus l/aspartate. Phos- phate concentrations: 0, 0.1 M, 0.2 M, and 0.3 M. Sodium gIycyI- glycine, 0.2 M, pH 7.8, at 28”, with 2pg per ml of catalytic subunit.

L I I I I ' 2468

I I I I

(I/&; (tnM)-i i8 24

FIG. 6. Product inhibition by phosphate, varying carbamyl-P (CAP) at constant aspartate (1 X 10-4~). l/Vversus l/carbamyl- P. Phosphate concentrations: 2 X 10-5, 4 X 10-3, 1.33 X 10-2, 2.67 X KY, and 4 X lo+ M. Sodium glycylglycine, 0.2 M, pH 7.8, at 28”, with 2 pg per ml of catalytic subunit.

the concentrations of Substrate A and Product & are varied (Fig. 6), for which a competitive pattern is expected (20). The experimental results support all of the predicted patterns except where Substrate B (aspartate) and Product P (carbamylaspartate) are varied; in this case, competitive instead of noncompetitive inhibition is observed (Fig. 8). The experiment was performed four times. Noncompetitive inhibition requires that the common intersection be displaced from the ordinate. The conditions for the experiment shown in Fig. 8 were adjusted 0’ I I I I I I

to detect this displacement as sensitively as possible: aspartate 0 25 100 125

concentrations were varied to a very high level (0.04 M) to ap- & (M)I:

preach the extrapolated intersection as closely as possible, and FIG. 8. Product inhibition by carbamylaspartate, varying

the concentrations of carbamylaspartate were varied over a very aspartate at constant carbamyl-P (0.02 M). l/V versus l/aspar-

wide range (0 to 0.15 M) to achieve more widely divergent lines tate. Carbamylaspartate concentrations: 0, 0.05 M, 0.10 M, and

and thus to define the intersection as well as possible. 0.15 M. Sodium gIycyIgIycine, 0.2 M, pH 7.8, at 28”, with 2 pg per ml of catalytic subunit.


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From the experimental conditions and from the quality of the data, an upper limit for the magnitude of the very small displacement of the common intersection from the ordinate may be estimated as 5 M-‘. The inverse of this value, 0.2 M, is therefore a lower limit for

The rate constant for the forward chemical step, kg, cannot be insignificantly small compared to ke, since the equilibrium for the over-all reaction lies far toward the formation of carbamylaspartate. Therefore, k&, the dissociationconstant for aspartate from the ternary complex (Kib) must be appreciably greater than 0.2 M. This dissociation constant is considerably higher than the Ki for aspartate analogues (see below). The fact that binding of aspartate to the enzyme-carbamyl-P complex is so un- favorable suggests that some of the binding energy of n-aspartate may be used to facilitate the reaction with carbamyl-P. (For a recent discussion of the role of strain in enzymatic catalysis, see Jencks (22).) Additional support for this argument is found in an accompanying paper (4).

An experiment like that shown in Fig. 8 was also performed at pH 7.0 in 0.2 M imidazole acetate (4). At this lower pH, at which an analogue of aspartate such as succinate binds more strongly (see below), a common intersection displaced from the ordinate was observed, confirming the prediction for an ordered BiBi mechanism and yielding a value of 0.017 M as a lower limit for the dissociation constant of aspartate from the central complex at pH 7.0.

The scheme described above for determining kinetic parameters provides an excellent approximation of Ki,, but does not permit the direct determination of K,. However, the parameters obtained by fitting the product inhibition data from Figs. 7 and 8 to the appropriate kinetic equations may be used to cal- culate the value of the ratio, K&Ka. This value is 1.5 f 0.2, which substantiates the assumptions made in calculating V,,,, Kb, and K;, from the apparent kinetic parameters described by Equations 3a, 3b. and 4b.

Calculation of Rate Constants-For the mechanism represented in Fig. 9 and by Equation 2, the ratio V,,,/K, equals lcl, the rate constant for the binding of Substrate A (carbamyl-P). Taking the approximate value, K;JKo equals 1.5, at 28” and pH 7.8, kl equals 1.6 x lo* per set per molar enzyme (mol wt 100,000). (This rate constant should be divided by the number of active sites per catalytic subunit to give the constant for each site.)

The ratio Vmllx/&, equals

which must be less than k3. Thus the lower limit for kt, the rate constant for the binding of Substrate B (aspartate), equals 7.5 x lo4 per set per molar enzyme at 28” and pH 7.8.

Using nuclear magnetic resonance spectroscopy, Sykes, Schmidt, and Stark4 have determined rate constants for the binding of two analogues of aspartate at 28” and pH 7.0. In the presence of saturating carbamyl-P, the rate constants for the

4 B. D. Sykes, P. G. Schmidt, and G. R. Stark, unpublished results.

EAB

EQP k7

FIG. 9. Ordered BiBi mechanism for the catalytic subunit of aspartate transcarbamylase. A, first substrate (carbamyl-P); B, second substrate (aspartate); P, first product (carbamylaspartate); &, second product (phosphate); E, enzyme.

binding of succinate and of n-malate are both on the order of lo5 per set per molar enzyme. These analogues have very different dissociation constants (see below), but their binding rates are similar and are strikingly slow.

If the rate for binding of aspartate is also slow, then the calculated lower limit for k~ would actually be a fair approximation for ka. Therefore, it seems likely that the rate constant for binding aspartate (k3) is orders of magnitude smaller than the rate constant for binding carbamyl-P (ICI). A very slow rate for binding aspartate is consistent with a conformational change during the binding process. Collins and Stark (4) have observed a large ultraviolet difference spectrum upon binding of succinate to the enzyme-carbamyl-P complex, which suggests a conformational change during the binding process. It seems not unlikely that the over-all rate may be limited by the rate of the step in which aspartate is bound.

Nonlinear E$ects-Replots of the slopes of the experiments shown in Figs. 5 and 6 plotted against the concentration of inhibitor are distinctly nonlinear (Figs. 10 and 11). The parabolic

I I I I I I

Ca~bamylaspat-rate (mM)

FIG. 10. Replot of slopes from Fig. 5 (l/V versus l/carbamyl-P) versus carbamylaspartate concentrations. Same conditions as for Fig. 5.


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FIG. 11. Replot of slopes from Fig. 6 (l/V versus l/carbamyl-P (CAP)) versus phosphate concentration. Same conditions as for Fig. 6.

EP WE- P

EQ,- -EQB Q II

I B

EQB FIG. 12. Ordered BiBi mechanism for aspartate transcarbam-

ylase, showing dead end pathways. Same symbols as for Fig. 9.

competitive inhibition of carbamyl-P by phosphate (Figs. 6 and 11) indicates a dead end branch from the reaction pathway. The only species which can provide parabolic inhibition by phosphate without disrupting the competitive pattern is E&z: that is, a ‘2nd mole of phosphate binds to the enzyme, possibly in the carbamylaspartate sit.e (Fig. 12). Since both substrates and both products are dianions, it seems possible that their binding sites might be somewhat similar. The data for the product inhibition experiment shown in Fig. 6 were fitted with a computer program with the use of the equation describing parabolic competitive inhibition:

v = vmx s

Km I2

1++,+-- +s (5)

1, KiK<<

The two dissociation constants for phosphate, K+, for dissociation of phosphate from the EQ complex (from the first order inhibition constant, Ki), and &, for the dissociation of phosphate from the ‘<wrong” site in the EQ2 complex (from the second order inhibition constant, Ki;), are Ki, = 1.4 f 0.1 x 10e3 M, and KS = 4.4 f 1.0 X 10e2 M. When a similar experiment was performed at pH 7.0, the values for the two dissociation constants were Ki, = 1.2 X low3 M and KS = 4.1 X low2 M (both corrected for ionization).

Since phosphate can bind in the wrong dianion site, carbamyl- P might also bind in this site, giving rise to an IL42 complex (Fig. 12). I f such a complex exists, substrate inhibition is predicted for carbamyl-P, because an A2 term is introduced into the denominator of the full rate equation. To test this, the concentration of carbamyl-P was varied up to an extremely high value (0.12 M), while aspartate concentration was held constant at a rate-limiting level (1OV M). The data were fitted to Equation 6, which describes substrate inhibition, and the results are summarized in Table II.

v = vnmx x

Km + S + X2/& (6)

The existence of EQ2 and EA2 complexes suggests the possible existence of E&A and EAQ complexes. However, an E&A complex, with carbamyl-P bound to the carbamylaspartate site of the EQ complex, is unlikely, because if carbamyl-P concentration were high enough to promote significant binding to the wrong site then it would compete with the phosphate to form EA rather than EQ. An EAQ complex would cause noncompetitive inhibition of carbamyl-P by phosphate, since an A& term is introduced into the denominator of the full rate equation. However, as shown in Fig. 6, competitive inhibition is observed, indicating that an EAQ complex, if present, does not contribute significantly to the kinetics.

An E&B complex (Fig. 12) has been observed by Collins and Stark (4), using difference spectroscopy. Such a complex would cause substrate inhibition by aspartate, because an AB2 term is introduced into thedenominator of the full rate equation. To test this possibility kinetically, the concentration of aspartate was varied up to an extremely high value (0.12 M) while the concentration of carbamyl-P was held at a constant, saturating level

TABLE II Substrate inhibition by carbamyl phosphate

Assay conditions were 2 pg per ml of catalytic subunit, rate- limiting aspartate (10m5 M), 0.2 M sodium giycyigiycine, pH 7.8, and 28”. V,,,,=, 620 ymoles per min per g; Km = 1.4 X 10e6 M;

Ki = 7.3 X 1P M.

Carbamyl-P V

M pmoles/min/g

3.0 x 10-s 115 8.0 X 1O-6 234 1.5 x 10-S 359 8.0 X lo+ 532 3.2 X lo-“ 598

1.25 X 1OP 609 1.0 x 10-z 518 4.0 x 10-Q 401 1.2 x 10-1 274


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(0.01 M). The results, shown as a reciprocal plot in Fig. 13, clearly reveal deviation from linearity at very high aspartate concentrations, and confirm the predicted substrate inhibition by aspartate. The data were fitted with the computer program for Equation 6.

Since the value of Ki is so close to the value of Kb, determination of Kb is unreliable unless the complete substrate inhibition experiment is performed. The values given in Table I are the results of such experiments at pH 6.4, 7.0, 7.8, and 8.7. The observed Ki for substrate inhibition by aspartat,e at saturating carbamyl-P equals

where Kp is the constant for dissociation of aspartate from the E&B complex, and the rate constants are those shown in Fig. 9. Since some of the terms within the parentheses are likely to be much greater than 1, Kz is actually much less than the observed Ki. A direct determination of K* at pH 7 has been made by Collins and Stark (4) using ultraviolet difference spectroscopy.

Dicarboxylate anions bind weakly to the free enzyme, much more weakly than to the enzyme-carbamyl-P complex (4). For this reason, high concentrations of carbamylaspartate lead to the formation of another dead end species, a weak EP complex (Fig. 12). This species is responsible for the nonlinear replot of the slopes from Fig. 5 versus carbamylaspartate concentration, shown in Fig. 10. An EP complex would not cause the replot of the intercepts from FI,. ‘0. 5 to be nonlinear, and in fact the replot is linear. The replot of slopes (Fig. 10) indicates a value of about 0.1 M for Kb, the constant for the dissociation of carbamylaspartate from the EP complex.

Aspartate also binds weakly to the free enzyme, giving an EB complex (4). However, compe&ive inhibition of carbamyl-P by phosphate (Fig. 6) indicates that any contribution of a dead end EB complex or of an alternate reaction pathway in which B binds first is very small compared to the major pathway, in which A binds first. The ordered addition of substrates and dissociation of products in the main reaction pathway and the existence of dead end enzyme-aspartate (EB) and enzyme-carbamylaspartate (EP) complexes indicate that carba.myl-P cannot bind to the EB complex and that phosphate cannot bind to the EP complex. A reasonable physical model, consistent with the catalytic mechanism proposed by Collins and Stark (4), is that, in the EB complex, the a-amino group of L-aspartate blocks the carbamyl-P-binding site; similarly, in the EP complex, the oc-ureido group of carbamyl-L-aspartate blocks the phosphate- binding site.

Tests for Carbamyl-Enzyme Intermediate-Phosphate is a competitive inhibitor for carbamyl-P (Fig. 6), and therefore the formation of a carbamy-enzyme intermediate with a dissociable phosphate cannot occur. This result rules out the possibility of a ping pong3 mechanism and is consistent with the observation of Reichard and Hanshoff (23) that native aspartate transcarbamylase will not catalyze the exchange of phosphate between carbamyl-P and 32P-orthophosphate. Since the formation of a carbamyl-enzyme might conceivably depend upon the binding of the second substrate, we looked for exchange of 32P-orthophosphate into carbamyl-P in the presence of succinate and the catalytic subunit, according to the procedure of Reichard and Hanshoff (23). In either the presence or the absence of succi-

nate, incorporation of 32P into carbamyl-P is about 0.2% after 10 min at an enzyme concentration of 65 pg per ml, which agrees almost exactly with the value obtained by the previous workers without enzyme. The low level of exchange in the absence of enzyme can be accounted for by the reversible dissociation of carbamyl-P to cyanate and phosphate. The possibility remains that formation of a carbamyl-enzyme might require the binding of the actual second substrate rather than an unreactive analogue, but the competitive inhibition observed indicates that, if such a hypothetical carbamyl-enzyme intermediate is formed, phosphate cannot dissociate from it before the remaining steps in the reaction occur.

Substrate Activity of CAP Analogues-Of six analogues tested, only acetyl phosphate and N-methylcarbamyl phosphate func- tioned in an enzyme-catalyzed reaction with aspartate. Methyl phosphate, ethyl phosphate, N-(p-nitrophenyl)carbamyl phosphate, and N, N-dimethylcarbamyl phosphate gave no evidence for the formation of the appropriate alkyl or acyl derivative of L-

aspartate in the presence of excess aspartic acid, even with a large amount of enzyme. The ability of these last four analogues to function as substrates was tested by determining the amount of aspartate remaining in each reaction mixture by amino acid analysis rather than by using the assay with radioactive aspartate, since the potential products would not be expected to pass through a Dowex 50 column without retention. Catalytic subunit (0.375 mg per ml), aspartate (2 X lo+ M), and analogue (2.5 x 1O-2 M) were incubated at pH 8 and 28’. The

I 50

I I I 100 200

i/Asp (M)+

FIG. 13. Substrate inhibition by aspartate. l/V versus l/ aspartate. Sodium glycylglycine, 0.2 M, pH 7.8, at 28”, with 0.01 M carbamyl-P and 2pg per ml of catalytic subunit. -, expected curve for simple Michaelis-Menten kinetics; - - -, expected curve for kinetics with substrate inhibition.


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TABLE III Kinetic parameters for analogues of carbamyl-P

Assay conditions were 0.2 M sodium glycylglycine, pH 7.8, at 28”. Values for Kb, Ki (for aspartate inhibition), and V,,, were determined at a saturating concentration of carbamyl-P analogue (0.01 M). Values for Ki, were determined at rate- limiting aspartate (10F6 M for carbamyl-P, 1OW M for acetyl phosphate, and 5 X lo-* M for N-methylcarbamyl phosphate). Con- centrations of catalytic subunit used were 2 pg per ml for carbamyl-P, 20 pg per ml for acetyl phosphate, and 143 pg per ml for N-methylcarbamyl phosphate.

&a Kb Ki VKLUX

______

!JM 11zM ‘u mole/min/g

Carbamyl-P. . . . . 14 20 0.17 0.90 Acetyl phosphate. . . . 490 21 0.43 2.2 x 10-Z N-Methylcarbamyl

phosphate. . . . . . . . 60 140 0.76 2.7 X 1O-4

Estimated error (%) . f10 f10 f20 f10

0.6

0.4

$0.2

6 c 0 .- E >.O> a,

z

=-.02

.Oi

C

I I I I

I I I I

I I I I

I 6

I I I 7 a 9

PH FIG. 14. pH dependence of catalytic subunit. Upper, with

carbamyl-P (0.01 M) and 2 pg per ml of catalytic subunit; Zower, with acetyl phosphate (0.01 M) and 20 Pg per ml of catalytic subunit. Imidazole acetate, 0.2 M, below pH 7.5; 0.2 M sodium giycyl- glycine above pH 7.5. Temperature 28”, with 0.04 M aspartate.

amount of aspartate did not decrease significantly (~2%) in 6 hours.

Kinetics and pH Dependence with Acetyl Phosphate-Grisolia, Amelunxen, and Raijman (24) showed that crude extracts of E. coli could catalyze the concurrent disappearance of acetyl phosphate and aspartate and concluded that the bacterial aspartate transcarbamylase could utilize acetyl phosphate in place of carbamyl-P. This conclusion is borne out by our present work with the purified catalytic subunit.

Kinetic experiments with acetyl phosphate were performed in the same way as those described for carbamyl-P, at 28” and at pH 7.8 in 0.2 M sodium glycylglycine. Since the enzymatic reaction is much slower with acetyl phosphate than with carbamyl- P, a higher enzyme concentration (20 pg per ml) was used. The results are shown in Table III.

Because the reaction is slow, it was also possible to repeat the determination of the apparent K, for acetyl phosphate (and N- methylcarbamyl phosphate, see below) at a higher aspartate concentration (0.04 M) to confirm that K, is not very different from Ka (see Equation 4b).

At pH 7.8, reaction with acetyl phosphate occurs with a maximum velocity 2.4% of that with carbamyl-P, and the K;, for acetyl phosphate is about 35-fold higher. Although the substi- tution of CHs for NH2 has a substantial effect both on affinity and reaction rate, the fact that acetyl phosphate does function as a substrate is an important piece of evidence in formulating a mechanism of action for aspartate transcarbamylase, because it shows that the NH2 group of carbamyl-P is not absolutely required for function.

The pH dependence of the catalytic subunit with carbamyl-P may best be seen from the values of the kinetic parameters at different pH values, over the range pH 6.4 to 8.7, as shown in Table I. The pH dependence of the initial rate of reaction at a single assay condition with acetyl phosphate or carbamyl-P is shown in Fig. 14. The data indicate that the pH dependence is far less sharp for acetyl phosphate than for carbamyl-P, and that the apparent optimum lies below pH 7 rather than near pH 8. The slower reaction with acetyl phosphate may have a different rate-limiting step, so that its pH dependence reflects the pK, values of different side chains or enzyme-substrate intermediates.

Kinetics with N-Methylcarbamyl Phosphate-N-Methylcar- bamyl phosphate is an extremely poor substrate for the catalytic subunit, with maximum velocity about 0.03% of that of carbamyl-P at pH 7.8. Since the rate is so slow, it is important to show that the reaction is in fact catalyzed by the enzyme. In the presence of catalytic subunit (1.3 mg per ml) and excess (5 x lop2 M) 14C-aspartate, an amount of radioactive N-methylcarbamylaspartate equivalent to 90% of the initial amount of N-methylcarbamyl phosphate (1 x lop2 M) was formed in 24 hours at 28”; the conversion increased to 105% in 72 hours. In the absence of enzyme, 2yo and 6% of the initial amount of N- methylcarbamyl phosphate appeared as radioactivity not re- tained by Dowex 50 after 24 and 72 hours. Since the reaction times were long, the small amount of apparent carbamylation in the absence of enzyme may well have been due to degradation of aspartate by bacteria in the reaction mixture.

Kinetic experiments with N-methylcarbamyl phosphate were performed in the same way as those with carbamyl-P and acetyl phosphate. Since the enzymatic reaction with N-methylcarbamyl phosphate is very much slower than with carbamyl-P or acetyl phosphate, a much higher enzyme concentration (143 pg per ml) was used. The results are shown in Table III.

There is no reason to expect on mechanistic grounds that sub- stitution of the nitrogen of carbamyl-P with a single methyl group would decrease the maximum velocity of the reaction SO

drastically. Therefore, it seems reasonable to conclude that N- methylcarbamyl phosphate is bound to the catalytic subunit predominantly in an unproductive mode and that N , N-dimethylcarbamyl phosphate, which is not a substrate, is bound exclu-


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sively so. For methyl groups to have such a large effect, the fit of the amide end of carbamyl-P into its binding site must be extremely tight. On the other hand, evidence presented below suggests strongly that the phosphate group is bound to a freely accessible site.

TABLE IV Constants at pH 8 and 18” for dissociation of carbamyl phosphate

and its analogues from catalytic subunit

cOmpOuna Ki

Inhibition by Analogues of Carbamyl-P-The data summarized in Table IV were obtained, for the most part, by studying the effect of various concentrations of inhibitors on the rate of the reaction at constant concentrations of both substrates. The concentration of carbamyl-P was 1 x lop3 M (about 70 times Ki, at pH 8) and the concentration of L-aspartate was about one- tenth of &,.5 The inhibition by analogues of carbamyl-P is linear, competitive. In a few cases, complete plots of 1 /V against l/S at a series of inhibitor concentrations were made; these yielded a common intersection on the ordinate, and replots of slopes against inhibitor concentrations were linear. For the most part, the Ki values in Table IV were obtained from linear plots of l/V against inhibitor concentration. The initial slopes of plots of product concentration against time and the plots of l/V against inhibitor concentration were calculated with a computer, with least squares programs. Since carbamyl-P, acetyl phosphate, and N-methylcarbamyl phosphate are substrates, the K; values shown in Table IV were taken from the kinetic data described above.

Carbamyl phosphate. ..................... Inorganic phosphate. ..................... Pyrophosphate ........................... Acetyl phosphate. ........................ AT-Methylcarbamyl phosphate ............. N,N-Dimethylcarbamyl phosphate. ....... N-(p-Nitrophenyl)carbamyl phosphate. ... Methyl phosphate. ....................... Ethyl phosphate .......................... CTP ..................................... Phosphonacetic acid. ..................... Phosphonacetamide ....................... N-Methylphosphonacetamide ............. N-(p-Nitrophenyl)phosphonacetamide ..... Methyl phosphonate. .....................

?nM 0.014 1.45 0.09 0.49 0.06 0.15 0.12 0.82 0.76 0.37 0.32 0.66 1.54 0.10 0.78

TABLE V

Inhibition of catalytic subunit by analogues of L-aspartate, $8”

Kleppe (16) has already reported that several inorganic anions, among them pyrophosphate, phosphate, and sulfate, are competitive inhibitors of the native enzyme and of native enzyme that has had its regulatory capability destroyed by heating. With the exception of carbamyl-P, which has a much lower dissociation constant than any other compound in Table I, the affinity of inhibitors for the catalytic subunit varies only by about a factor of 25. No compound containing a phosphate or phosphonate dianion failed to inhibit the enzyme, indicating that the phosphate-binding site of the enzyme is readily accessible. The similarity of dissociation constants in Table IV indicates that the rest of the inhibitor or substrate molecule contributes little to the energy of binding. Binding does seem t.o be tighter for carbamyl-P and N-methylcarbamyl phosphate and for those inhibitors which carry a large hydrophobic group (the two p-nitrophenyl compounds).

Analogue PH Ki

Changeux, Gerhart, and Schachman (21) have noted that two classes of binding sites for CTP are seen in equilibrium dialysis experiments with native aspartate transcarbamylase: a set of sites with high affinity, consistent with binding to the regulatory sites, and an additional set of sites with much lower affinity. Gerhart and Schachman (2) had previously found that 5-bromo- CTP binds to more than the expected number of regulatory sites at saturation of the native enzyme. In view of our observation that CTP is a good competitive inhibitor for carbamyl-P, it seems clear that these results can be explained by the binding of CTP both to the CTP sites on the regulatory subunit and to the carbamyl-P sites on the catalytic subunit.

Succinate 6.0 Succinate 6.9 Succinate 7.9 Succinate 8.3 Succinate 8.6 Succinate 8.8 L-Malate 6.9 L-Malate 7.9 L-Malate 8.8 n-Malate 6.9 Malonate 7.9 Glutarate 7.9 Maleate 7.8 Fumarate 7.8 n-Aspartate 7.9 L-Bromosuccinatec 6.9 n-Bromosuccinate” 6.9

m&l

0.44a 0.87 3.5 9.3

19.6 20.5 12.1 61

144 11.5 27

260 4.3

145 No inhibition*

70 107

5 Corrected for incomplete ionization of succinate at this pH; pK, = 5.6 was used for the second dissociation.

*The concentration of L-aspartate was 0.01 M and that of D-

aspartate was 0.12 M. c Sodium cacodylate buffer was used, to avoid reaction of the

inhibitor with imidazole or glycylglycine.

6 From Equation 4b it can be seen that when B = [aspartate] = 0.1 K*,

apparent K, = 1OKia + Ka 11

Inhibition by Analogues of Aspartate-The experiments were carried out in the same way as those described for analogues of carbamyl-P, and the results are summarized in Table V. The constants reported in Table V were determined in the presence of saturating carbamyl-P and refer to dissociation of the analogue from the catalytic subunit-carbamyl-P complex. Inhibi- tion by analogues of aspartate is linear, competitive. In some cases, KG values were calculated from plots of 1 /V against inhibitor concentrations; in others, the concentration of aspartate was varied at constant concentration of inhibitor, the data were fitted to a hyperbola as described above, and the KS was calcu-

Since K, is not very different from Ki,, Ki, is an excellent approximation for apparent K, under the conditions used for determination of KS values for analogues of carbamyl-P. Therefore Kd, has been used in these calculations.


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.020- P &.016-

10-'/H

FIG. 15. pH dependence of apparent Kd for succinate. Ki (apparent) versus l/H. Data of Table V have been used.

lated from the apparent K,. Results for the same inhibitor by the two methods were in good agreement, and the Ki calculated did not depend upon the concentration of inhibitor used.

Succinate and maleate are the best inhibitors among those tested; it is already known from the work of Gerhart and Pardee (25) that these two compounds inhibit the native enzyme effi- ciently as well. The constant determined by competitive inhibition for dissociation of succinate from the catalytic subunit agrees well with those determined by equilibrium dialysis for the native enzyme and catalytic subunit (21). Malonate is much less effective than succinate as an inhibitor, and glutarate is extremely poor, indicating that a good fit of a 4-carbon dicarboxylic acid into the binding site for aspartate is important. In agreement with unpublished results for the native enzyme,B fumarate is not a good inhibitor of the catalytic subunit, indicating that aspartate and succinate bind in conformations that must resemble closely the cis-configuration of maleate. D-AS- partate (0.12 hr) does not inhibit the catalytic subunit at pH 8, in agreement with the results of Reichard and Hanshoff (23) for the native enzyme. However, both D- and L-malate are inhibitors and, surprisingly, they have similar dissociation constants. D- and L-Bromosuccinate also inhibit the enzyme competitively. With much longer times of exposure than those necessary for a kinetic experiment, these reactive analogues of aspartate inac- tivate the catalytic subunit.7 Gregory and Wilson (26) have shown that bromosuccinate inactivates native aspartate transcarbamylase at a much more rapid rate than the catalytic subunit.

The data indicate that any dicarboxylic acid larger than succinate binds less tightly than succinate to the catalytic subunit. Any 4.carbon dicarboxylic acid with an Lu-substituent binds less tightly than succinate. In fact, even the dissociation constant for the natural substrate, L-aspartate, is much higher than the constant for succinate. These results suggest that any cr-substituent interferes sterically with the tight binding of dicarboxylic acids. Since the cr-substituent of the actual substrate (the amino group of aspartate) is the site for reaction, it seems possible that the mode of steric interference is that the cY-substituent is forced into close contact with the carbonyl group of carbamyl-P, favoring the chemical reaction. For additional support for this proposed mechanism, see the accompanying paper by Collins and Stark (4).

6 J. C. Gerhart, personal communication. 7 G. L. Herve and G. R. Stark, unpublished results.

Dependence of Inhibition by Substrate Analogues on pH-The constant for dissociation of carbamyl-P from its binary complex with enzyme, Ki,, is not greatly dependent upon pH between pH 6.4 and pH 8.7. Similarly, competitive inhibition by phosphonacetamide* is virtually independent of pH over the same range. Since both of these compounds are dianions, they are probably bound to positively charged groups on the enzyme. The pH independence observed suggests that side chains of ly- sine or arginine are probably involved, since they do not lose their positive charge at pH 8.7.

In contrast, as can be seen from the data of Table V, inhibition of the catalytic subunits by succinates and by n-malate is strongly dependent upon pH. The fact that affinity decreases with increasing pH indicates that a positively charged group on the enzyme that is critical for binding becomes discharged upon upward titration. The pK, for this functional group has been calculated from the data for inhibition by succinate by using the equations for linked functions described by Edsall and Wyman (28). The equilibria and dissociation constants are

EH

E EHI

K a

= (E) (HI EH

K, = (EH) (1) z EHI

K, = @I) (H) a EHI

K!, = (-79 (0 1 EI

where E is the concentration of free enzyme (uncomplexed with I and unprotonated on the functional group that aids in the binding of I), H is the concentration of hydrogen ion, I is the concentration of inhibitor, and EH, EI, and EHI are the concentrations of the complexes. The apparent Ki at any pH is given by Equation 7.

ICE + EH) K; (apparent) = EI + EHI (7)

Substituting from the above definitions of dissociation constants and rearranging yields Equation 8.

Kg (apparent) = :‘i-+ Ka) --+”

a (8)

When the data for inhibition by succinate are plotted as Ki (apparent) against l/H (Fig. 15), a linear plot is obtained. Equation 8 reduces to a linear function of l/H only if K,K</ K’< << H. (If KaKi/K’i were >>H, then Ki (apparent) would be a linear function of H, which is not true.) Therefore, when H is of the order of K,, K’i >> Ki, which agrees with the observation that the inhibitor is bound much less strongly to the un-

8 The concentrations of the dianions of each inhibitor have been determined by correcting the molar concentration for incomplete ionization at the lower pH values, with pK, 6.5 for phosphonacetamide (determined titrimetrically at 28”) and pK, 5.6 for succinate (27).


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Issue of April 10, 1969 R. W. Poyter, M. 0. Modebe, and G. R. Stark 1859

protonated form of the enzyme. Neglecting the term K,Ki/ 4. K’i, Equation 8 reduces to

5.

K; (apparent) = Ki + T

In this equation, the ratio of slope to intercept equals K,. Linearity of the plot in Fig. 15 also indicates that only a

single functional group is being titrated, since a sharper dependence on pH than that observed would be expected if two or more positively charged groups were being discharged over the same range. The pK,, calculated from the limiting intercept and a least squares best fit for the slope of the curve, is 7.1 s 0.1 for a group, perhaps an imidazolium ion, that must be positively charged for binding of succinate to the enzyme-carbamyl-P complex to occur. Any other positively charged groups involved in the binding of succinate do not titrate in the pH range investigated.

From Equation 8 it can be seen that, when H >> K,, Ki (apparent) = Ki, so that the limiting value of Ki is given by the

y-intercept; this value is 4.4 X lop4 M for succinate.

7.

8.

9. 10. 11.

CRAMER, F., AND WEIMANN, G., Chem. Ber., 94, 996 (1961). CRAMER, F., AND WINTER, M., Chem. Ber., 92, 2761 (1959). ALLEN, C. M., JR., RICHELSON, E., AND JONES, M. E., inN. 0.

KAPLAN AND E. P. KENNEDY (Editors), Current aspects of biochemical energetics, Academic Press, New York, 1966, p. 401.

12. 13.

HOLMBERG, B., Chem. Ber., 60, 2198 (1927). HUNNINGHAKE, D., AND GRISOLIA, S., Anal. Biochem., 16,

200 (1966). 14. STARK, G. R., AND SMYTH, D. G., J. Biol. Chem., 238,214 (1963). 15. BRESNICK, E., AND Mossk., H., Biochem. J., 101, 63 (1966). 16. KLEPPE, K., Biochim. Biophys. Acta, 122, 450 (1966). 17. LINK, T. P., AND STARK, G. R., J. Biol. Chem., 243, 1082 (1968). 18. CLELAND, W. W., Advan. Enzymol., 29, 1 (1967). 19. BETHELL, M. R.,.SMITH, K. E:, WHITE, J. S., AND JONES, M.

E.. Proc. Nat. Acad. Sci. U. S. A.. 60. 1442 (1968). Acknowledgments-We are most grateful to Dr. John Gerhart for providing us with samples of enzyme that were useful in early iy: exploratory experiments, for stocks of E. co% defective in oro- tidylate decarboxylase, and for several stimulating discussions. 2% We would also like to acknowledge the generous gifts from the Macy Foundation and the National Institutes of Health which 23, made possible the use of the Advanced Computer for Medical Research at the Stanford Medical Center. 24.

1.

2.

3.

REFERENCES 25. 26.

GERHBRT, J. C., AND PARDEE, A. B., J. Biol. Chem., 237, 891 27. (1962).

GERHART, J. C., AND SCHACHMAN, H. K., Biochemistry, 4, 1054 (1965).

GRISOLIA, S., AMELUNXEN, R., AND RAIJMAN, L., Biochem. Biophys. Res. Commun., 11, 75 (1963).

GERHART, J. C., AND PARDEE, A. B., Fed. Proc., 23, 727 (1964). GREGORY, D. S., AND WILSON, I. B., Fed. Proc., 27, 785 (1968). HODGMAN, C. D., WEAST, R. C., SHANKLAND, R. S., AND SELBY,

S. M. (Editors), Handbook of chemistry and physics, The Chemical Rubber Publishing Company, Cleveland, 1962, p. 1756.

SCHMIDT, P. G., STARK, G. R., AND BIILDESCHWIELER, J. D., 28. J. Biol. Chem., 244, 1860 (1969).

EDSALL, J. T., AND WYMAN, J., Biophysical chemistry, VoZ. I, Academic Press, New York, 1958, p. 653.

COLLINS, K. D., AND STARK, G. R., J. Biol. Chem., 244, 1869 (1969).

GERHART, J. C., AND HOLOUBEK, H., J. Biol. Chem., 242, 2886 (1967).

SPECTOR, L., JONES, M. E., AND LIPMANN, F., in S. P. COLO- WICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. 111, Academic Press, New York, 1957, p. 653.

FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem., 66, 375 (1925).

BALSIGER, R. W., JONES, D. G., AND MONTGOMERY, J. A., J. Org. Chem., 24, 434 (1959).

CLE&ND, W. W., Biochim. Biophys. Aka, 67‘, 104 (1963). CHANGEUX, J.-P., GERHART, J. C., AND SCHACHMAN, H. K.,

Biochemistry, ‘7, 531 (1968). JENCKS, W. P., inN. 0. KAPLAN AND E. P. KENNEDY (Editors),

Current aspects of biochemical energetics, Academic Press, New York, 1966, p. 273.

REICHARD, P., AND HANSHOFF, G., Acta Chem. &and., 10, 548 (1956).


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Robert W. Porter, Michael O. Modebe and George R. StarkSUBUNIT

Aspartate Transcarbamylase: KINETIC STUDIES OF THE CATALYTIC

1969, 244:1846-1859.J. Biol. Chem.

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