THE JOURNAI. OF BIOLOG~CAL CHEMISTRY Vol. 255. No. I O . I s u e of May 2.5. pp 4ii2-4iHo. 1980 Prcnted ~n '. S. A.

(Received for publication, November 13, 1979)

David J. T. Porter and Harold J. Bright From the Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

We describe the interactions at 25°C of the 3-carb- anions (I-) and 3-carbon conjugate acids (I) of 3-nitro- propionate, 3-nitro-2-hydroxypropionate, and 3-nitro- 2-aminopropionate with fumarase and aspartase.

H X H X I I

Ia-, X = H I I

@ I I I OzN"C"C"C02- 02N-C-C"COz- Ib-, X = OH

H H H IC-, X = NH2 I- I

1. Ia and Ib inhibit fumarase competitively and are bound more than 4000- and 18,000-fold more tightly, respectively, than Ia and Ib, and 5000- and 11,000-fold more tightly, respectively, than succinate. IC inhibits aspartase competitively and is bound (depending on the ionization state of the 2-amino group) more than 220- to 1630-fold more tightly than IC and 290- to 2200- fold more tightly than succinate.

2. Fumarase and aspartase bind I- much more tightly than their respective substrates. Thus, K,/K,, and K J K s for fumarase are 375 and 900, respectively (with KI, = 64 nm and K s = 27 1 1 ~ at pH 7.0). For aspartase, K,/KI, = 519, while K,/KI, = 1630 (2-amino group unprotonated) or 220 (2-amino group proton- ated).

3. The values of k1 and k-1 (E + I- e E - -I-) for

fumarase are, respectively, 0.55 X 10' M-' s" and 3.5 s-' for Iii and 2.6 X 10' M-' s-' and 6.9 s-' for 15 at pH 7.0,

These results, together with those of control experi- ments with Malic Enzyme, suggest that Ia, 16, and IC are transition state, or transient intermediate, ana- logues and that the mechanisms of the fumarase and aspartase reactions involve enzyme-bound 3-carban- ions.

k, k-I

Fumarase (EC 4.2.1.2) and aspartase (EC 4.3.1.1) catalyze the reversible elimination of H20 from L-malate and NHa from L-aspartate, respectively (Equation 1).

c0,- c02- H I \ /

I - l l + HX (X = OH or NHd (1)

I / \ con- H c02-

H-C-H - C

H-C-X C

* This work was supported by Grant GM 11040, National Institutes of Health.

As the result of isotope effect studies (1-3), the fumarase reaction is often discussed in terms of a 2-carbonium ion (El ) mechanism. However, the timing of C-H and C-OH bond cleavage is difficult to assess from such studies (4, 5) and the %carbanion (EIcB) and concerted (E2) mechanisms are prob- ably not ruled out by the available evidence. Less work has been done with aspartase and both carbonium (5) and carb- anion (6) mechanisms have been considered. A 3-carbanion intermediate has been proposed in the related 3-methylaspar- tase reaction (7).

The use of transition state analogues, the basis for which was first stated by Pauling (8 ) , provides an alternative ap- proach to the question of the sequence of covalent changes undergone by enzyme-bound substrate along the reaction coordinate (9-13). Simple transition state theory predicts that the enzyme should bind a transition state many orders of magnitude more tightly than the ground state substrate from which it is derived. In practice, binding enhancement factors tend to be in the range from lo2- t,o 105-fold and most com- pounds so tested are likely to be analogues of metastable intermediates which resemble a transition state derived from the intermediate more closely than they resemble the sub- strate (10, 14).

With these considerations in mind we have tested 3-nitro- propionate (Ia, X = H), 3-nitro-2-hydroxypropionate (Ib, X = OH) and 3-nitro-2-aminopropionate (or 3-nitroalanine, IC, X = NH,) as inhibitors of fumarase and aspartase.' These isosteric and isoelectronic analogues are carbon acids (pK = 7.75 to 9.7) which, in their resonance-stabilized conjugate base (carbanion) states IS, Ib, and IC, closely resemble the nitronate valence bond structure (15, 16, and Equation 2). They may therefore be used as transition state, or transient intermediate, analogues to test the hypothesis that these enzymes generate 3-carbanions from their substrates during catalysis.

As a control experiment, we examined the inhibition of malic enzyme (EC 1.1.1.40) by Ib. Although the mechanism of this reaction may well involve a 3-carbanion intermediate (17), this would be the enolate of pyruvate resulting from decarboxylation rather than proton abstraction. Thus, Ib would be a poor analogue for this intermediate carbanion and I6 would not be expected to be a better inhibitor than Ib.

A previous report from this laboratory (18) described the inhibition of mitochondrial succinic dehydrogenase by 15. However, this inhibition is irreversible and may well involve an oxidation-reduction mechanism. The carbanion 1% is there-

' The abbreviations and notations used are: Ia, Ib, and IC, tee conjugate 3-C acids of the nitroalkanes defined in Equation 2; IZ, Ib, and IC, the conjugate 3-C bases (3-carbanions) of Ia, Ib, and IC; Hdc, HIE, IcH-, and IC*-, the microscopic ionization states of IC defmed in Equation 8 Ia,eq, Ib,eq and Ic,eq, solutions of Ia, Ib, and IC which are protonically equilibrated at 3-C.

4772

Carbanion Binding to Fumarase and Aspartase 4773

0 , - , o - , o N

I

I + // N+-0" Ia, X = H

o- 1 I b , X = O H I

H-C-X I co,

I I

fore probably not functioning solely as a transition state, or metastable intermediate, analogue in this reaction (18, 19).

EXPERIMENTAL PROCEDURES

Materials-Fumarase (porcine heart), aspartase (Bacterium ca- daueris), and Malic Enzyme (chicken liver), each of the highest purity offered by Sigma, were used without further purification and were treated as described in the Sigma technical bulletin. L-Malate, L- aspartate, and NADP were also from Sigma. ~~-3-Nitro-2-hydroxy- propionate (Ib) was synthesized as described by Shechter and Conrad (20) (m.p. = 75-76OC, 75-76OC reported in Ref. 20). 3-Nitropropionate (Ia) was from Aldrich and was used without further purification because results obtained with it and with sublimed material (gift of Theodore Alston of this laboratory) were indistinguishable. 3-Nitroa- crylic acid was synthesized as described by Shaw (21) (m.p. = 133- 134"C, 134-136°C reported in Ref. 21). ~~-3-Nitro-2-aminopropionate (IC) was obtained by treating DL-3-

nitro-2-chloropropionate, synthesized as described by Shaw (21) and Shechter et al. (22). with ammonia. T o 3 g of ~~-3-nitro-2-chloropro- pionic acid was added 35 ml of concentrated NHIOH dissolved in 150 ml of methanol. The reaction was allowed to proceed for 2 h under argon at 25°C and the solution (which had slowly turned brown) was then taken to dryness on a rotary evaporator a t 20°C. The black residue was dissolved in -15 ml of water, the pH was adjusted to 4.5 with HCI, and the solution was then treated with Norit a t 3OoC for 5 min and filtered. Following the addition of 2 volumes of isopropyl alcohol crystallization occurred overnight a t 0°C. Recrystallization from ethanol/water (1:l) at 0°C gave white needles, with an overall yield of 61.3% (m.p. = 152-153°C with decomposition)

Calculated: C 26.87, H 4.51, N 20.89, 0 47.43 Found: C 26.58, H 4.55, N 20.91, 0 47.48

and 60 MHz NMR spectroscopy (0.5 M IC in 1 M 2HC1 and 'H20, 3- (Trimethylsily1)-1-propanesulfonic acid as standard) showed 6 5.32 (doublet, J = 1.6 Hz), 6 5.24 (singlet), and 6 4.83 (apparent multiplet). Titration yielded an equivalent molecular weight of 130 (calculated, 134).

Methods-Fumarase was assayed spectrophotometrically through fumarate production from L-malate a t 210 to 240 nm. Assay solutions were buffered with 0.05 M potassium phosphate between pH 6.0 and 7.5, with 0.05 M Tris/acetate between pH 7.5 and 9.5 and with 0.05 M triethanolamine-HCI at pH 10.0. When the K,,, for malate in the absence of buffer effects was required, 0.01 M Tris/acetate at pH 7.4 was used.

Aspartase was assayed similarly through fumarate production from L-aspartate at 210 to 240 nm in solutions containing 0.1 M Tris/ acetate, 2 mM MgS04, and 0.1 M EDTA at all pH values examined. Concentrations of K' a t a given pH value were maintained constant with KC1 but varied over the pH range studied owing to changes in the range of potassium aspartate required.

When fumarase and aspartase were assayed in the presence of carbanionic inhibitors undergoing protonic equilibration the monitor- ing wavelength was 215 nm because this is an isosbestic point for the conjugate carbon acid and base forms of these compounds.

(23). Malic enzyme was assayed according to the method of Hsu et al.

Equilibrium mixtures (Ia,eq, Ib,eq, and Ic,eq) of the conjugate carbon acid and base states of Ia, Ib, and IC were obtained by incubating the carbon acid at the desired pH until the absorbance a t 240 nm was invariant. Solutions of the pure carbanions IS, 16, and IC were prepared as described previously (24). Concentrations of the carbanions were determined spectrophotometrically at X,,, values of

nitronate valence bond structure

231 nm (15, E = 9,700 M" cm"), 242 nm (16, e = 9,360 M" cm-l) and 241 nm (I?, e = 9,230 M" cm").

Spectral and slow kinetic measurements were carried out on Gil- ford, Cary 14 and Cary 118 spectrophotometers, while rapid kinetic measurements were performed on the Gibson-Dunum stopped flow spectrophotometer. All experiments were conducted at 25°C unless otherwise noted. When a transformation of data to a linear form was not possible the data were fitted using a weighted less squares program written by Dr. Carl Fuller of this laboratory following the procedure described by Wentworth (25).

RESULTS

Studies of Fumarase Stability a n d Properties of 3-Nitropropionate a n d 3-Nitro-

2-hydroxypropionate-There are several plausible mecha- nisms for the nonenzymic breakdown of Ib. Using the criteria of the stability of X,,, at 242 nm and the time independence of emax we found I6 to be completely stable in 1.0 N KOH for at least 2 h. In particular, nitromethane anion (which is unstable under these conditions, but which can be monitored at its X,,, = 231 nm as it breaks down) was not produced. Equilibration of the conjugate carbon acid of Ib in 0.1 M sodium pyrophosphate at pH 8.5 resulted in a slow absorbance increase at 242 nm (t% =. 150 s) after which X,,, and ~ 2 ~ 2 were stable for 2 h. Again, there was no evidence for the formation of nitromethane. The absorbance transient is due to conver- sion of 10% of Ib to I6 at this pH value.

Fig. 1 shows that the rate of protonation of I5 a t 3-C at pH 7.0 corresponds to k = 1.67 X s-' while that for 16 is k = 2.75 X 10" s-' at pH 7.5.15, but not 16, is therefore sufficiently stable to be used directly as a preformed 3-carbanion in initial steady state velocity measurements without significant inter- ference from its conjugate 3-carbon acid. The inset of Fig. 1 represents spectrophotometric titrations of the 3-carbon acid functions of Ia and Ib (each point representing the pH attained in a separate solution of Ia or Ib after equilibration for 1.0 h following the addition of KOH) and gives 3-carbon acid pK, values of 9.0 and 9.7 for Ia and Ib, respectively.

Inhibition of Fumarase by L -3-Nitro-2-Hydroxypropio- nate-We measured the inhibition of fumarase in the pH range from 6.0 to 10.0 by Ib,eq (see "Methods"). Since the pK, value for the carboxyl group of Ib is less than 3.8 (25), such solutions contain the dianionic (carbanionic) and mon- oanionic species of Ib but insignificant amounts of the neutral molecule. Using L-malate as variable substrate, we found Ib,eq to give linear competitive inhibition across the entire pH range. These data are reported in Fig. 2 as K , / K I , ~ ~ . We express the results in this way because the K , values for L- malate are very dependent on pH and on the ionic composition of the assay medium (27, 28) and we expect such effects to cancel in the ratio K,,, /KI, ,~. The data of Fig. 2 fit the titration curve of a monobasic acid with pK, = 9.7 and a limiting, pH- independent, value for K,,,/KI,,,, (= K,,,/KIG) of 900. Since the pK, for ionization of Ib at 3-C is also 9.7 (see previous section),

4774 Carbanion Binding to Fumarase and Aspartase

I I

C - li 0 2

‘241 (IC1

0 . 0 ~ “ ” ’ ” ” ’ 200 400 600 800 1000

Time (sed

FIG. 1. Rates of protonation at carbon of the preformed 3- carbanions of 3-nitropropionate (la), ~~-3-nitro-2-hydroxy- propionate (Zb), and ~~-3-nitro-2-aminopropionate (IC, de- scending curve), as well as the rate of deprotonation at 3-C of the conjugate carbon acid of ~b3-nitro-2-aminopropionate (IC, ascending curve). The experimental conditions, which were chosen to be identical with those used for subsequent enzymic studies, were 0.01 M Tris/acetate, pH 7.0, for Ia; 0.025 M Tris/acetate, pH 7.5, for Ib; and 2.0 m MgSO,, 0.1 mM EDTA, 0.05 M Tris/acetate, pH 8.5, for IC. The concentrations of the nitroalkanes were 0.092 m in each case and the reactions were monitored spectrophotometrically at 241 nm and 25°C.

I O(

i .o 8.0 10.0 12. pH

FIG. 2. The pH dependence of K,,,/K,I,,~ for the binding of protonically equilibrated mixtures of ~b3-nitro-2-hydroxy- propionate to fumarase at 25°C. It is assumed that only the 2 - ~ isomers of Ib,eq bind to the enzyme. The solid line represents the titration of a monobasic acid with pK = 9.7 and an amplitude of 900. Reaction conditions are described under “Methods” and enzyme concentrations varied between 50 and 400 ng/ml, depending on the pH.

the data of Fig. 1 are interpreted to mean that I6 binds 900 times more tightly than L-malate to fumarase and that Ib binds no more than about one-twentieth as well as L-malate. The values of K,,,/Kr- are compiled in Table I, where, it should be noted, we assume that only the 2 - ~ isomers of Ib,eq bind to the enzyme. The observed K,,, values for L-malate varied between 0.81 rn (pH 6.6) and 5.54 mM (pH 10.0).

This interpretation of the results of Fig. 2 can be tested through the addition of preformed 16 to the assay system at pH 7.5. At this pH, which is over 2 units below pK, = 9.7, the 3-carbanion will reprotonate almost completely at a rate de- termined by k = 2.75 X lo-’ s-’ (see previous section). Thus, a lag in fumarate production is predicted if 16 is the inhibitory species, while a burst is expected if Ib is inhibitory and neither a lag nor a burst if I6 and Ib bind equally well.

3-carbanic Enzyme (variabh

substrate)

TABLE I The inhibition of fumarase, aspartase, and malic enzyme by

tn analogues and their conjugate acids at 25’ C e

Fumaraseb (L-Malate)

Aspartaseb (L-Aspartate)

Malic enzymeb @-Malate)

“K,istheM

Inhibitor I K J K I I K J K I “

3-Nitropropionate (Ia) L-3-Nitro-2-hydroxypropionate (Ib) Succinate 3-Nitropropionate (Ia) L-3-Nitro-2-aminopropionate (IC)‘

H ~ c HIc- IcH- ICz-

Succinate L-3-Nitro-2-hydroxypropionate (Ib)

~

375 900

519

220 1630

~0.05

t l . O

(Kdd 0.95 t15‘ (Ki)d 1.54 t15‘

:haelis constant for the variable substrate; KI- is the 1 [ic

inhibition constant for (where appropriate, the 2 - ~ isomer of) the 3- carbanion of the nitroalkane inhibitor; KI, similarly, is the inhibition constant for (where appropriate, the 2 - ~ isomer of) the conjugate carbon acid of the inhibitor.

Reaction conditions given under “Methods.” Enzyme concentra- tions were, depending on the pH, 50 to 400 ng/ml (fumarase), 0.05 to 0.25 units/ml (aspartase), and 0.56 pg/ml (Malic Enzyme).

Ionic species of IC defined by Equation 8. See Equation 11.

‘It is assumed that it is oossible to observe a 0.3-fold increase in this ratio when the pH is changed from 7.0 to 8.0.

FIG. 3. Lags in fumarase activity caused by the ad-dition of the 3-carbanion of ~~-3-nitro-2-hydroxypropionate (Ib) and its subsequent protonic equilibration. All solutions contained 5.0 mM L-malate, 0.025 M Tris/acetate and fumarase (85 ng/ml) at pH 7.5 and 25°C and fumarate was monitored at 215 nm, which is an isosbestic point for the conjugate carbon and acid states of Ib. The lag times (ti, see Equation 3) were 85 s and 110 s for 88 p~ and 176 p~ carbanion, respectively, and were also functions of the concentra- tion of L-malate and the rate of reprotonation of the carbanion. At this pH approximately 0.5% of Ib is present as the inhibitory 2-~-3- carbanion when protonic equilibration is completed after about 200 s.

The traces of Fig. 3 clearly show a lag in fumarase activity following the addition of IL and they are most simply modeled by Scheme I, from which we derive the extent of the lag (tl)

as Equation 3.

E + S - E . S + E + P Ks k:!

Carbanion Binding to Fumarase and Aspartase 4775

This treatment assumes, justifiably, that the pH is sufficiently less than the pK, of the 3-carbanion (I-) that reprotonation of the latter is irreversible. From the experimental values of tl in Fig. 3 we calculate values of lo00 and 1110 for KS/KIK with 0.088 mM and 0.176 m~ I6 initially, respectively. These ratios are very sensitive to tl, the value of which is inherently inaccurate. Nevertheless, the average ratio of 1055 is in quite good agreement with the value of 900 computed forK,/KIi; from the equilibrium data of Fig. 2 and we may conclude that both sets of experiments agree in showing that the 3-carbanion of the 2 - ~ isomer of Ib binds approximately lo00 times more tightly than L-malate to fumarase and that the monoanionic (conjugate carbon acid) 2 - ~ isomer of Ib, at best, binds only one-twentieth as well as L-malate.

Inhibition of Fumarate by 3-Nitropropionate-Solutions of Ia,eq also caused linear competitive inhibition of fumarase with L-malate as variable substrate. The data obtained at pH 7.8 and 25°C in 0.01 M Tris/acetate, gave K, , , /KI~.~~ = 18.4. If we assume that the only significantly inhibitory species is the 3-carbanion (pK, = 9.0) then KJKE = 310.

However, in this case the 3-carbanion reprotonates suffi- ciently slowly ( 4 2 % protonation after 60 s at pH 7.0, see Fig. 1) that it can be directly evaluated as a competitive inhibitor if initial steady state velocities of fumarate formation are measured within 60 s or so of the initiation of the reaction. From such experiments at pH 7.0 and 25°C in 0.01 M Tris/ acetate, we calculated K, = 24 PM for L-malate (which agrees well with the value given in Ref. 29) and KE = 64 n ~ . Thus, K,,,/KE = 375, which agrees satisfactorily with the value of 310 calculated from the ratio K ~ / K I . ~ ~ and pK, = 9.0. These results not only confirm directly that the inhibitory species is the 3-carbanion but also validate the procedure of expressing inhibition as the ratio K,,,/KI,~~ Thus, the K,,, value for L- malate at pH 7.8 is over 200 times larger than that obtained at pH 7.0, while the value of K,,,/K& calculated from K,/KI,~~ and pK, = 9.0 for the 3-carbanion is only 17% smaller than the value of KJK, obtained directly from the experiments using preformed IS.

Rates of Dissociation of Carbanionic Inhibitors from Fu- marase-When the enzyme is converted to E -1- by the ad- dition of 1% or I6 and E * I- is then mixed with L-malate in the stopped flow spectrophotometer, a lag in fumarate production is observed from which the rate of dissociation of the E .I- complex may be calculated. An example of such a reaction trace is given in the inset of Fig. 4. Provided that [SJ/K,,, >> [I-]/KI- the dissociation of E.1- is essentially irreversible because free E is immediately trapped as E a S and this exper- iment is then most simply described by Scheme 11.

E.I-%E + I- E + S / E * S - E + P Ks kz

SCHEME I1

The time dependence of fumarate formation is given by Equa- tion 4.

while the expression for the linear extrapolation shown in the inset of Fig. 4 is given by Equation 5.

0 . 3 1 -

0.2 sec

0.0 1 , " 0.0 0.2 0 .4 0.6 0.8 1.0

Time (sec)

FIG. 4. The rates of dissociation of the 3-carbanions of 3- nitropropionate (Ia) and 3-nitro-2-hydroxypropionate (16) from the E*T- complex of fumarase, measured in 0.01 M "is/ acetate at pH 7.0 and 25°C and plotted according to Equation 6. E.15 (generated from 1.0 p~ I5 and 32 p g / d of fumarase) was mixed with an equal volume of 5.0 m~ L-malate and fumarate was monitored at 240 nm. The stopped flow trace from this experiment, together with that from a contr_ol experiment in which I5 was omitted,

this complex was generated from 0.2 m~ protonically equilibrated appears in the inset. The E.Ib experiment was similar, except that

Ib,eq and 17.6 p g / d of fumarase.

where t' represents time ir, the linear extrapolation. The value of k-I can be determined either by setting P(t ' ) = 0 in Equation 5 or subtracting Equation 5 from Equation 4 to give Equation 6 and then taking the slopes of semilogarithmic plots of Equation 6.

-k , t A t = t' - t =e

k-r (6)

The data of Fig. 4, when plotted semilogarithmically ac- cording to Equation 6, yield values for k-1 of 3.5 s-' and 6.9 s" for IZ and 16, respectively. Since the KI- (= k-t/kI) values for 1% and I6 under these conditions (low buffer concentration) were 64 nM and 27 nM, respectively, the values of k~ are 5.5 X lo7 M" s-' for 15 and 2.6 X 10' M-' s-' for 16. These values approach the on rate for L-malate measured by Alberty and Pierce (30).

Are 3-Nitro-2-Hydroxypropionate and 3-Nitroacrylate Substrates for Fumarase?-If nitroacrylate and Ib are sub- strates for fumarase, the catalyzed reaction would be that of Equation 7.

OH H I I

I / H H

Hz0 + -OzC"CH=CH"NOz + -0zC- C -C"NOz (7)

With fumarate and malate the equilibrium lies appreciably to the right because A G O = -0.85 kcal mol" (6) and the equilib- rium of Equation 7 would be expected to be even further to the right because of the nitro group. Consequently, we tested nitroacrylate at pH 7.0 in 0.05 M potassium phosphate and found that, within the limit of detection (= 0.1% of the activity of L-malate), it was inactive as a substrate. Nitroacrylate was also not an inhibitor of malate consumption (K,/KI < 0.1). Furthermore, no loss of activity was observed when 2 mM nitroacrylate and 0.8 n" Ib,eq were incubated with fumarase at pH 7.4 for 15 min and 60 min, respectively. Thus, although nitroacrylate is substantially electrophilic and, apart from net charge (31), resembles fumarate closely, there is no evidence

that it can complex with, or alkylate an essential residue of, the enzyme.

Studies of Aspartase Stability and Properties of DL -3-Nitro-2-Aminopropio-

nate"Severa1 nonenzymic mechanisms for the breakdown of IC can be envisaged, including the nonoxidative 2,3-elimina- tion of ammonia to form nitroacrylate as has been shown to slowly occur with 3-nitro substituted cyclic amino acids (32) as well as the nonoxidative 2,3-elimination of nitromethane utilizing nonbonding electrons of the 2-amino group. Conse- quently, the stability of IC was examined. No changes in X,,, (241 nm) or emax occurred after 2 h incubation of IC in 1.0 N KOH at 0°C. When placed in 0.05 M Tris/acetate, pH 8.6 at 25"C, containing 2 mM MgS04 and 0.1 m EDTA to simulate the assay conditions for aspartase, the absorbance of IC reached the same value at 241 nm as that of a second sample in H20 alone at its isoelectric point. However, a slow ( t% = 7 h) decrease in Az4,, accompanied by an increase in A313, was noted in the pH 8.6 buffer solution and the solution gradually became brown in color. Thus, IC is fairly stable and solutions of it at pH 8.6 are suitable for enzymic experiments when used within 2 h of their preparation.

The equilibration of the 3-carbanion of Ic with the conjugate acid is shown in Fig. 1. The relaxation times and position of equilibrium are independent of the initial state, as expected, and this information is used in the following section.

The Microscopic p K , Values of DL -3-Nitro-2-Aminopropi- onate-The acid-base equilibria of IC are described by four microscopic pK values when fractional protonation of the carboxylate is negligible (Equation 8).

NH?

II

K 1 11"' NH?

I

H

Solution of this system requires the evaluation of one micro- scopic constant and two macroscopic constants ( K A = K l K d ( K , + K 2 ) , KB = K3 + K4) or two macroscopic constants and the sum ([IC2-] + [IcH-I). All three items can be evaluated in this system.

The value of pK4 was determined by titrating the isoelectric species (i.e. HJc) with KOH. The equilibration of HJc and HIE with IcH- and IC" after titration is sufficiently slow (t% = 60 s from A241 measurements as shown in Fig. 1) that pH measurements made within 10 s of the addition of KOH reflect the isolated equilibrium of K4. These data are sum- marized in Table I1 and give pK4 = 7.31. The two macroscopic constants were obtained from the titration curve of Fig. 5. In this case complete equilibration of all four species of Equation 8 was required and the time interval between successive additions of KOH was therefore 3 to 10 min. The data of Fig. 5 were fitted to pKA = 9.40 and pKB = 7.17 and to amplitudes corresponding to an equivalent molecular weight of 130 (cal- culated, 134). The microscopic dissociation constants of Equa- tion 8 are therefore pK1 = 9.27, pK:! = 8.82, pK3 = 7.75 and p& = 7.31.

TABLE I1 Macroscopic and microscopic acid dissociation constants for DL -3- nitro-2-aminopropionate*(see Equation 8) at 25°C in 0.1 M KC1

Dissociation constant Value

4776 Carbanion Binding to Fumarase and Aspartase

I K4 I (HIE) O~N-CHL--C--CO~-; H+ r 02N"CHz"C"CO2F (HJc) I

9.40" 7.17" 9.27h 8.82h 7.75h 7.31'

" Obtained as best fits to the titration curve of Fig. 5. Obtained from the relationships K A = KIK2/(K1 + K2) , KH = K:I

+ Kq and KAKB = K1K4 = K ~ K J . e Obtained by titrating four 1.0-ml aliquots of IC at its isoelectric

point (each containing 35.6 peq IC in 0.1 M KC1 and having initial pH values of 4.96, 4.95, 4.96, and 4.96, respectively) with 5.0, 10.0, 15.0, and 20.0 peq. KOH (added as 1.0 N KOH). The pH values obtained within 10 s of the addition of KOH were 6.53, 6.91, 7.21, and 7.45, respectively, giving calculated p& values of 7.31, 7.30, 7.33 and 7.32, respectively. See "Results" for the rationale of this method.

These values can be checked independently by comparing the calculated and observed fractions of 3-carbanion, ([IC"] + [1cH-])/[Ic]~, as a function of pH. This comparison is shown in the inset of Fig. 5, where it is assumed that the €241 values for IC'- and IcH- are identical. Despite the fact that the observed fraction of 3-carbanion was slightly sensitive to ionic strength (which was not closely controlled in the deter- minations of the pK values), the agreement between the calculated and observed quantities of 3-carbanion is excellent.

Inhibition ofAspartase by L -3-Nitro-2-Aminopropionate- The initial steady state velocity patterns for aspartase show

I H

I H

slight positive cooperativity (33, 34). However, over carefully chosen ranges of L-aspartate concentration, the double recip- rocal plots are substantially linear and solutions of Ic,eq exhibited linear competitive inhibition with K , / K I , , ~ = 116 at pH 8.0. The K' concentration varied from experiment to experiment (5 to 100 mM) but was held constant at a given pH. It was assumed that K,,,/K,., would be K'-independent even though K,,, and are not.

The question of whether the inhibitor is (HIE + H21c) or (IC'- + IcH-) is answered by the experiments of Fig. 6. Here we note a lag in fumarate production when (IC'- + IcH-) is added but a burst when (HIE + H21c) is added, the initial velocity of which is identical with that observed in the absence of inhibitor. These results are those expected if IC" or IcH-, or both, are inhibitory, if neither HIE nor HJc is inhibitory and if, as we have already demonstrated in Fig. 1, these pairs of species slowly equilibrate protonically.

The pH dependence of K , , , / K I , ~ ~ shown in Fig. 7 (where we have assumed that only the 2 - ~ isomers of Ic,eq bind to the enzyme) does not fit the titration curve of a monobasic acid (note the dashed line of slope +1 in the log plot of Fig. 7) and

Carbanion Binding to Fumarase and Aspartase 4777

,OOt pK, ~ 9 . 4 0

200 / pKB=7.17

0.0

pH

5 7 9 I1 13 I I I I I

PH

FIG. 5. Titration of 3-nitro-2-aminopropionate (IC) with KOH at 25°C to obtain the macroscopic constants ~ K A and pKe. A solution of IC (20.0 mg in 3.0 ml of 0.1 M KC1) was titrated with 10-p eq aliquots of KOH and the pH was read after each addition of KOH when ionization of the carbon acid function had come to equilibrium (see “Results”). The inset shows the observed (experi- mental points) and calculated (solid line) fraction of 3-carbanion of IC ( f i - ) , as a function of pH, when 0.2 m~ IC was titrated in 0.1 M Tris/acetate buffer solutions. The 3-carbanion was monitored spec- trophotometrically at 241 nm.

Time (sed

FIG. 6. Demonstration that the 3-carbanion of 3-nitro-2- aminopropionate is the inhibitor of aspartase. The traces rep- resent fumarate production monitored spectrophotometrically at 215

2.0 mM MgS04, 0.1 mM EDTA, and 0.05 unit of aspartase/ml at pH nm in solutions containing 2.5 mM t-aspartate, 0.05 M Tris/acetate,

8.5 and 25’C. The top trace represents the reaction in the absence of IC. The middle trace results from the addition of 0.13 m~ IC as the conjugate acid (HIE + Hdc, see Equation 8) while the bottom trace results from the addition of 0.093 mM IS as the preformed 3-carbanion (IC*- + IcH-, see Equation 8).

suggests, instead, that either IC” and IcH-, or HIE and H ~ I c , are competitive inhibitors of L-aspartate and that the mem- bers of the active pair have different Kr values. Thus, taken together, the experiments of Figs. 6 and 7 identify Ic2- and IcH- as the competitive inhibitors of L-aspartate, at least in the concentration ranges tested. This being so, the data of Fig. 7 can be expressed as Equation 9, where fie^- and f i c ~ - repre- sent the fractions of total nitroalanine present as IcH- and IC’-, respectively.

Using the microscopic dissociation constants for Equation 8 to generate f i c ~ - and fz- the best fit of the data of Fig. 7 yields K,,,/Kl,2 = 1630 and K,,,/K,<n- = 220. The final form of Equa- tion 9 is given by Equation 10, from which the solid line in Fig. 7 was calculated

K , 3.9 X lo-‘’ [H’] + 4.4 X -=

[H’]’ + 6.8 X lo-“ [H’] + 2.7 X 10”’ (10)

The scatter in the experimental points of Fig. 7 is due to the limited range of L-aspartate concentrations over which linear double reciprocal plots could be obtained and to the large K,, values for L-aspartate at the higher pH values. The observed K,,, values for L-aspartate varied from 0.78 mM (pH 6.0) to 80 m~ (pH 9.5).

Inhibition of Aspartase by 3-Nitropropionate-Ia,eq was a linear competitive inhibitor of aspartase with L-aspartate as variable substrate and at pH 8.0 the value of K,,,/K1,ec, was 53. Assuming that, as with IC, it is the 3-carbanion which is responsible, we calculate K,,,/Krl = 519. Under the same con- ditions succinate gave K,,,/KI = 0.8. Thus, the amino group of IcH- and IC” (protonated or unprotonated) does not appear to be important in determining the value of K I - while the nitronate function (see Equation 2), compared to a carboxyl- ate, is of great significance.

Are 3-Nitro-2-Aminopropionate and 3-Nitroacfylate Sub- strates for Aspartuse?-It was not possible to determine whether aspartase catalyzed the thermodynamically favorable (Keq < 0.5 mM at pH 7.0) addition of ammonia to nitroacrylate because the nonenzymic reaction is itself quite rapid (tlr2 = 260 s at pH 7.0 with 0.05 M NH,Cl and 0.05 M potassium phosphate). Furthermore, 0.073 mM nitroacrylate inactivated the enzyme to the extent of 60% in 5 min a t pH 7.4 (0.05 M Tris/acetate, 2 mM MgS04 and 0.1 m~ EDTA).

Consequently, we assayed aspartase in the unfavorable direction of 2,3 elimination of ammonia from Ic,eq by deter- mining whether the enzyme became inactivated. We found no

1

pH 8 IO

FIG. 7. The pH dependence of K,, , /KI~,~, , for the binding of protonically equilibrated mixtures of DL-3-nitrO-2-aminOprO- pionate (Ic,eq) to aspartase at 25°C. It is assumed that only the 2 - ~ isomer of IC binds to the enzyme. The solid line was calculated from Equation 10 as described in the text. Reaction conditions are given under “Methods” and aspartase concentrations varied between 0.05 and 0.25 unit/&. The dashed line represents the slope expected if only one of the 3-carbanionic species, IC” or IcH- (see Equation 8), binds to the enzyme.

4778 Carbanion Binding to Fumarase and Aspartase

TABLE I11 Steady state parameters for inhibition of malic enzyme by 3-nitro-

2-hydroxypropwnate at 25°C The data, analyzed according to Equation 11, were obtained from

initial steady state velocities of NADPH formation (monitored at 340 n m ) in solutions containing 0.1 mM NADP, 0.05 M triethanolamine- HCl, 40 mM MgC12,0.067 mg/ml of bovine serum albumin, and 0.56 g/ml of malic enzyme. Only the 2 - ~ isomers of Ib, eq were assumed to bind to the enzyme.

Parameter uH 7.0 DH 8.0 M M

K , (L-malate) 8.3 X 10-~ 3.8 X

K1.q 8.7 X 5.6 X 10" Ki.q 5.4 X 2.2 X 10" KnJK1.q 0.95 0.68 K J K L 1.54 1.72

loss of aspartase activity after the enzyme had been incubated for 87 min at 0°C in 2 m~ Ic,eq at pH 8.0 (in a solution containing 0.1 M Tris/acetate, 2 m~ MgSO., and 0.1 mM EDTA). Thus, we could find no evidence that IC is a substrate for aspartase.

Inhibition of Malic Enzyme by 3-Nitro-2-hydroxypropio- nate-We examined the interaction of Ib with malic enzyme because I6 should be a poor transition state analogue for this reaction (17) and is therefore not expected to bind with greater affiiity than Ib or L-malate itself. The addition of Ib,eq at pH 7.0 and 8.0 resulted in noncompetitive inhibition patterns when L-malate was the variable substrate. These patterns were analyzed according to Equation 11.

The values of K,, KI,eq, and Ki.eq are given in Table I. Although a detailed analysis of KrSeq and Kt,, was not carried out, the pH independence of K,,,/Kr,,q and of K,,,/K;,eq in the range from 7.0 to 8.0 shows that it is the conjugate 3-carbon acid state of Ib, and not the 3-carbanion, which is functioning as the inhibitor. If 16 were an inhibitor the values of Km/Ki,eq for this species would have to be much less than 15. Thus, in contrast to fumarase and aspartase, the inhibition of malic enzyme by the nitro analogue is relatively weakly noncom- petitive and the inhibitory species is the conjugate carbon acid.

The addition of Ib,eq (1.0 m ~ ) in place of L-malate in the standard assay system resulted in no detectable reduction of NADP. As with fumarase and aspartase, therefore, the nitro analogue does not appear to be a substrate for malic enzyme.

DISCUSSION

Throughout these studies we have used K,,, as a measure of substrate binding and have expressed the affinity of inhibitors relative to that of substrate as KJinhibition constant. This is common practice in papers dealing with transition state ana- logues even though nonbranched uni-substrate reactions (with which class many of the studies of transition state analogues have been concerned) generate K,,, values which may bear any numerical relationship to the substrate dissociation constant when two or more intermediates are involved. Fortunately, data for fumarase (at least at pH 7.0) and for aspartase are available to show that the K,,, values for L-malate (30) and L- aspartate (33), respectively, are good estimates of the sub- strate dissociation constants. A second assumption concerned with the use of KJinhibition constant is that whatever changes occur in K,,, as the result of changes in reaction conditions (particularly pH, but including in addition specific

ion effects and ionic strength) will be reflected also in the inhibition constant, so that the ratio will not vary with the reaction conditions when the states of ionization of the sub- strate and inhibitor are fixed. This assumption was verified in the case of fumarase and 1% because, under conditions where the K,,, for L-malate varied over 200-fold, the ratio K,,,/Kr, varied only 17%.

The K,,,/Kr- and K,,,/KI values of Table I show that the 3- carbanions of the nitro analogues investigated are potent competitive inhibitors of fumarase and aspartase while their conjugate carbon acids are weakly (i.e. indetectably) inhibi- tory. The magnitudes of K,,,/KI- for the 3-carbanions compare favorably with those which have been obtained for other proposed transition state analogues and which are discussed in recent reviews (10-12). The carbanionic nature of 3-C, which appears to determine the strength of binding of the inhibitors, is expressed in aqueous solution as the nitronate valence bond structure form (15, 16).

0- 0- +/ /

R"CH=N R-CH=C

This is probably, then, the structural feature recognized by fumarase and aspartase and, except for charge, it is entirely analogous to the 3-carbanions of L-malate and L-aspartate stabilized over the carboxylate in the aci form (35). The importance of the nitronate form is emphasized by comparing the binding of Iii and succinate to the two enzymes. Although these molecules have the same net charge the carbanion is bound 4690 and 683 times more strongly than succinate by fumarase and aspartase, respectively.

The introduction of a hydroxyl group or an amino group into the 3-nitropropionate structure, in order to mimic fully malate and aspartate, respectively, causes only a 3-fold en- hancement of binding affinity of the 3 carbanions. This result suggests that these groups are of little importance in deter- mining the stability of the enzyme * inhibitor complexes and, by extrapolation, the stability of the respective carbanionic transition states in the catalyzed reactions. Furthermore, in the case of 3-nitro-2-aminopropionate and aspartase, the 3- carbanion having the protonated amino group (IcH-) binds 8 times less tightly than that with the unprotonated amino group (IC'-). If the 2,3 elimination of ammonia from L-aspar- tate involves a 3-carbanion intermediate then the leaving ammonia group should be protonated and it might be thought that IcH- would be a better transition state analogue than IC'-. However, protonation of the enzyme-bound amino group would be assured if the binding domain for the amino group involves an ionizable residue which, in its conjugate base state,

accepts the protonated amino group ( E + NH3-R) and in its

conjugate acid state the unprotonated amino group (EH + NH2-R). It should be noted that the pH dependence of K,/ KI,,q reflects ionizations of the free inhibitor (and free sub- strate) but is insensitive to ionizations of active site residues. It is also worth noting that the net charge on the transition state represented by an aspartate 3-carbanion with the amino group protonated is equivalent to the IC'-, rather than IcH-, state of 3-nitro-2-aminopropionate.

In contrast to fumarase and aspartase, malic enzyme was not preferentially inhibited by I6 but was moderately suscep- tible, in a noncompetitive fashion, to inhibition by the conju- gate carbon acid of Ib. Although the mechanism of this reaction probably involves a 3-carbanion intermediate,

+ +

Carbanion Binding to Fumarase and Aspartase 4779

namely, the enolate of pyruvate (17), neither this nor transi- tion states leading to it would be closely approximated by 16. The lack of inhibition by 16 presumably reflects this fact. It has been shown that carbonyl compounds bind to E. * NADPH and decrease the rate of reiease of NADPH (36). The fact that Ib was noncompetitive with L-malate suggests that Ib acts like a carbonyl compound and slows the release of NADPH from the enzyme.

We have assumed that the interactions between the carb- anions of Ia, Ib, and IC and fumarase and aspartase are entirely noncovalent. However, in addition to undergoing radical chain reactions (which are unlikely to have been involved in these studies), nitroalkane carbanions can function as nucleophilic donors in Michael and aldol reactions and, when suitably protonated, as acceptors in both nucleophilic and electrophilic addition reactions (37). Their capacity to react with nucleo- philes is of special concern because of the possibility that alkylation of an active site residue in fumarase or aspartase might be the reason, at least in part, for their very great affinity for these enzymes (Table I). The mechanism of the Nef reaction (38), which illustrates the principle of nucleo- philic addition, involves the attack of H20 at the carbon of

the fuUy protonated nitronic species ( fol-

lowed by elimination of cationic nitrogen to form a carbonyl compound (37). Although we cannot rule out an analogous reaction with an active site nucleophile with certainty, the following considerations make it unlikely. Firstly, the rate of combination of I6 with fumarase (2.6 X 10' M" s") is very similar to the rate of combination of L-malate with that enzyme (a process which almost certainly does not involve the formation of a covalent adduct) and is, likewise, close to the diffusion controlled limit for such bimolecular encounters (30). Thus, the free energy of activation for the reaction of I6 with fumarase is probably too small to accomodate the covalent processes involved in a Nef-type reaction and, furthermore, we have no evidence for biphasic inhibition of either fumarase or aspcrtase (which might correspond to the rapid formation of E -1- followed by the slower conversion of this to the covalent complex E-I). Similarly, the rapid, complete and uniphasic recovery of activity in Fig. 4, as well as the purely competitive inhibition patterns which were consistently ob- served in steady state experiments, are difficult to reconcile with a multistep covalent modification and its hydrolytic reversal. Secondly, the Nef reaction requires both oxygens of the nitronic group in the nitroalkane to be protonated ( e g . in 4 M HzS04) whereas IE, 16, and IE under our conditions, at least in the free state, have neither oxygen protonated. Thirdly, if I6 reversibly alkylates fumarase then it is surprising that 3-nitroacrylate (which is highly electrophilic and is re- lated to Ib, as is fumarate to malate, by the 2,3 elimination of HzO) does not inactivate fumarase through irreversible Mi- chael addition. Finally, the failure of I6 to inhibit malic enzyme, as well as the lack of reactivity of numerous nitrod- kane anions with active site amino acid residues of flavopro- tein oxidases (24, 39), suggests that these compounds, even when they are constructed to resemble physiological sub- strates, are not highly reactive as electrophiles toward active site residues.

We have used nitroalkanes extensively in previous studies as probes of the reaction mechanisms of flavoprotein oxidases (24, 39, 40). In those cases, however, they functioned as reactive substrates and we were able to obtain, through the use of external nucleophiles as trapping agents, direct evidence for carbanions and flavin-carbanion adducts as obligatory reaction intermediates in flavin reduction (24,39). We synthe- sized Ib and IC for the present studies on the supposition that

they, likewise, might be reactive substrates for fumarase and aspartase and that, additionally, they might generate 3-ni- troacrylate as an active site-directed alkylating agent following the 2,3 elimination of HzO or NH3. It is now clear that Ib and IC, in either their conjugate carbon acid or base states, are not substrates for fumarase and aspartase owing, presumably, to the fact that the carbanionic electrons in E . I5 and E .IE are so delocalized in the nitronate group as to lack the driving force for expulsion of OH- or NHs. The poor affinity of the conjugate carbon acids (Ia, Ib, and IC) for fumarase and aspartase (Table I) probably reflects the absence of a net negative charge at the zwitterionic nitro terminus of these ligands (31). In the case of mitochondrial succinic dehydro- genase, where we had reasoned that a flavin adduct derived from IS might spontaneously collapse to cause inactivation of the enzyme, it was found that IE, although not detectably oxidized, was indeed an irreversible inhibitor (18). The latter result was confi ied with purified, soluble, enzyme by Coles et al. (19) who suggested that IS or its oxidation product (3- nitroacrylate) reacts with an essential sulfhydryl group at the succinate binding site. In those studies, IE was stated to be oxidized by submitochondrial particles at about 0.1% of the rate given by succinate. However, since the enzyme was almost completely inhibited by -0.8 flavin equivalents of IS only a single enzyme turnover, approximately, should be observed. We should note also, in this regard, that the toxicity of Ia from natural sources such as the Indigo plant may in part be due to the inhibition of fumarase by Iii (Table I) as well as the inactivation of succinic dehydrogenase (18).

In conclusion, therefore, the major finding of these studies is that nitroalkane analogues of postulated 3-carbanion inter- mediates bind very strongly to fumarase and aspartase. We favor the interpretation that this behavior reflects the action of transition state, or metastable intermediate, analogues. This interpretation conflicts with proposals that these en- zymes catalyze a 2-carbonium ion mechanism (1,3,34). How- ever, the carbonium ion proposal rests largely upon secondary deuterium isotope effects and Rose (4) has suggested, in the case of fumarase, that these may also be explained through equilibrium isotope effects. Thus, a 3-carbanion mechanism would be consistent with the isotope effect data if, following abstraction from L-malate or L-aspartate, the 3-proton were shielded from solvent and if malate/fumarate and aspartate/ fumarate equilibria were established on the enzymes as the result of rate-determining product release. Recent studies of the pH dependence of primary and secondary isotope effects, as well as steady state parameters, in the case of fumarase have in fact given very strong evidence for a 3-carbanion mechanism (41). Furthermore, in collaborative studies follow- ing from those described herein, aconitase has been found to bind the carbanions of nitrocitrate and nitroisocitrate with dissociation constants as small as 680 PM (42). All of these results suggest that enzymes which abstract a proton from a highly basic sp3-hybridized carbon center may achieve this, in part, through binding-induced distortion of this carbon to a geometry resembling sp2 hybridization.

REFERENCES

1. Hansen, J. N., Dinovo, F. C., and Boyer, P. D. (1969) J. Biol.

2. Berman, K., Dinovo, F. C., and Boyer, P. D. (1971) Bioorg. Chem.

3. Schmidt, D. E., Jr., Nigh, W. G., Tamer, G., and Richards, J. H.

4. Rose, I. A. (1970) in The Enzymes (Boyer, P. D., ed) Vol. 2, 3rd

5. Klinman, J. P. (1977) Adu. Enzymol. 44,456-459 6. Hanson, K. R., and Havir, E. A. (1972) in The Enzymes (Boyer,

Chem. 244,6270-6279

1,234-242

(1969) J. Am. Chem. SOC. 91,5849-5854

Ed, pp. 281-320, Academic Press, New York

4780 Carbanion Binding to Fumarase and Aspartase

P. D., ed) Vol. 111, pp. 75-167, Academic Press, New York 7. Bright, H. (1964) J. Biol. Chern. 239,2307-2315 8. Pauling, L. (1946) Chem. Eng. News 24, 1375-1377 9. Lienhard, G. E., Secemshi, I. I., Koehler, K. A., and Linquist, R.

10. Lindquist, R. N. (1975) in Drug Design (Ariens, E. J. , ed) Vol. 5,

11. Wolfenden, R. L. (1976) Annu. Reu. Biochem. Bioeng. 5,271-306 12. Wolfenden, R. L. (1977) Methods Enzymol. 46, 15-28 13. Wolfenden, R. L. (1976) in Transition States of Biochemical

Processes (Gandour, R. D., and Schowen, R. L., eds) pp. 555- 578, Plenum Press, New York

N. (1971) Cold Spring Harbor Symp. Quant. Biol. 36,45-51

pp. 24-80, Academic Press, New York

14. Hammond, G. S . (1955) J . Am. Chem. Soc. 77,334-338 15. Jonathan, N. (1961) J. Mol. Spectrosc. 7, 105-115 16. Williams, F. T., Jr., Flanogan, P. W. K., Taylor, W. G., and

17. Schimerlik, M. I., and Cleland, W. W. (1977) Biochemistry 16,

18. Alston, T. A,, Mela, L., and Bright, H. J . (1977) Proc. Natl. Acad.

19. Coles, C. J., Edmondson, D. E., and Singer, T. P. (1979) J. Biol.

20. Shechter, H., and Conrad, F. (1953) J. Am. Chern. SOC. 75, 5610-

21. Shaw, P. D. (1967) Biochemistry 6, 2253-2260 22. Shechter, H., Conrad, F., Daulton, A. L., and Kaplan, R. B. (1952)

23. Hsu, R. Y., Lardy, H. A,, and Cleland. W. W. (1967) J. Biol.

24. Porter, D. J. T., Voet, J . G., and Bright, H. J . (1973) J. Biol.

25. Wentworth, W. E. (1965) J. Chem. Ed. 42,96-101 26. Sober, H. A,, ed (1968) Handbook of Biochemistry, p. 5154,

Shechter, H. (1965) J. Org. Chern. 30,2674-2678

576-583

Sci. U. S. A . 74,3767-3771

Chem. 254, 5161-5167

5613

J . Amer. Chem. SOC. 74,3052-3056

Chem. 242,5315-5322

Chern. 248,4400-4416

Chemical Rubber Co., Cleveland, Ohio

(1954) J. Am. Chem. SOC. 76,2485-2493

Chem. soc. 79, 1523-1525

868

1526-1530

27. Alberty, R. A,, Massey, V., Frieden, C., and Fuhlbrigge, A. R.

28. Frieden, C., Wolfe, R. G., Jr., and Alberty, R. A. (1957) J . Am.

29. Frieden, C., and Alberty, R. A. (1955) J. Biol. Chern. 212, 859-

30. Alberty, R. A., and Pierce, W. H. (1957) J. Am. Chern. SOC. 79,

31. Massey, V. (1953) Biochem. J . 55, 172-177 32. Burrows, B. F., and Turner, W. B. (1966) J. Chem. SOC. (Lond.)

33. Dougherty, T. B., Williams, V. R., and Younathan, E. S. (1972) Biochemistry 14,2493-2498

34. Williams, V. R., and Lartique, D. J. (1967) J. Biol. Chern. 242, 2973-2978

35. Pfeffer, P. E., and Silbert, L. S. (1971) J . Org. Chem. 36, 3290- 3293

36. Schimerlik, M. I., and Cleland, W. W. (1977) Biochemistry 16, 565-570

37. Nielsen, A. T. (1969) in The Chemistry ofthe Nitro and Nitroso Groups (Feuer, H., ed) Part 1, pp. 349-486, Interscience, New York

( C ) 255-260

38. Nef, J. U. (1894) Ann. Chem. 280, 263-291 39. Bright, H. J., and Porter, D. J . T. (1975) in The Enzymes (Boyer,

P. D., ed) 3rd Ed, Vol. 12, pp. 421-506, Academic Press, New York

40. Porter, D. J. T., and Bright, H. J. (1977) J. Biol. Chem. 252,

41. Blanchard, J. S., and Cleland, W. W. (1980) Biochemistry 19, in

42. Schhoss, J. V., Porter, D. J. T., Bright, H. J., and Cleland, W. W.

4361-4370

press

(1980) Biochemistry 19, in press