ARCHIVES
OF BIOCHEMISTRY
Initial Kinetic
AND
Characterization
PAUL Department
BIOPHYSICS
F. COOK
of Biochemistry,
178, 293-302
(1977)
of the Multienzyme Synthetase AND
RANDOLPH
University Received
of California, July
Complex,
Cysteine
T. WEDDING Riverside,
California
92502
13, 1976
Cysteine synthetase from Salmonella typhimurium LT-2 displays a saturation curve for sulfide identical to that obtained with uncomplexed 0-acetylserine sulthydrylase, indicating substrate inhibition with a K, of 0.1 5 0.017 mM and aK, of 0.303 2 0.194 mM. With both L-serine and acetyl CoA, however, cysteine synthetase exhibits two intermediary plateaus in the respective saturation curves. The time course of cysteine synthetase activity when the reaction is started by adding enzyme displays a pronounced lag phase. This lag is explained as being due to the buildup of a sufficient concentration of Oacetyl-L-serine to permit binding to 0-acetylserine sulfhydrylase. This conclusion is substantiated by the fact that plots of UT against concentrations of both L-serine and acetyl CoA reflect the saturation curves for these substrates. In addition, the incubation of the complex with L-serine and acetyl CoA results in the accumulation of the intermediate products of the reaction sequence, CoA and 0-acetyl-L-serine. Dissociation of the multienzyme complex under these conditions was ruled out by Sephadex G-200 chromatography of the complex after incubation with assay levels of the substrates of the reaction. Aggregation of cysteine synthetase was detected using disc gel electrophoresis and confirms earlier reports [Kredich, N. M., and Tomkins, G. M. (1966) J. Biol. Chem. 241,4955-49651. Aggregation of 0-acetylserine sulfhydrylase was also detected using the same technique.
The biosynthesis of L-cysteine in Escherichia coli and Salmonella typhimurium has been found to proceed via a two-step enzymatic pathway (1). Serine transacetylase is responsible for the conversion of acetyl CoA and L-serine to O-acetyl-L-serine and CoAl. The second step is catalyzed by 0-acetylserine sulfhydrylase, which converts 0-acetyl-L-serine and sulfide to Lcysteine and acetate. A physical association of serine transacetylase with 5% of the total cellular Oacetylserine sulfhydrylase has been reported by Kredich et al. (2). These investigators also reported resolution of this multienzyme complex by the product of serine transacetylase, 0-acetyl-L-serine, into one molecule of serine transacetylase (M, 160,000) and two molecules of O-acetylser1 Abbreviations used: DTNB, 5,5’-dithiobis (2-nitrobenzoic acid); OASS, 0-acetylserine sulfhydrylase; CoA, coenzyme A; OAS, 0-acetyl-L-serine. 293 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ine sulfhydrylase (M, 68,000) at concentrations above lop4 M. The turnover number for 0-acetylserine sulfhydrylase was found to double when this enzyme was resolved from serine transacetylase. Kredich et al. (2) also suggested that sulfide prevents dissociation of the complex ‘by O-acetyl-Lserine. The multienzyme complex has not been studied to date due to the lack of a sufficiently sensitive and rapid assay. The sulfide ion selective electrode allows the study of the overall reaction. These studies present an initial kinetic characterization of cysteine synthase. MATERIALS
AND
METHODS
Enzyme assays. Serine transacetylase was assayed by the method used by Kredich and Tomkins (1). A unit of transacetylase is defined as the amount of enzyme required to produce 1 ymol of thionitrobenzoic acid in 1 min at pH 7.6 and 25°C. Reactions were carried out in cells of l-cm path length in a final volume of 1 ml which contained:
294
COOK
AND
Tris-HCl, pH 7.6,0.1 M; DTNB, 1 mM; acetyl CoA, 2 mM; L-serine, 5 mM. The reaction was initiated by the addition of enzyme and the time course was monitored continuously with a recording Gilford Model 2400 spectrophotometer. The reaction was linear in all cases for at least 1 min. Rates were calculated using an extinction coefficient for thionitrobenzoic acid at 412 nm of 13,600 M-’ cm-‘. 0-Acetylserine sulfhydrylase was assayed with a sulfide ion selective electrode as described previously (3), using conditions for the standard assay. Initial velocities were also calculated as previously described. Cysteine synthetase was assayed using the sulfide ion selective electrode as described for O-acetylserine sulfhydrylase (3), continuously monitoring the disappearance of sulfide. All reactions were carried out at 25°C in a final volume of 1 ml. The standard assay contained Tris-HCl, pH 7.6, 0.1 M; acetyl CoA, 2 mM; L-serine, 5 mM; sulfide (added in 0.1 M Tris-HCl, pH 7.61, 0.2 mM; and was initiated with an appropriate enzyme aliquot. A point-by-point conversion of potential to sulfide concentration using the equation for the standard curve for sulfide obtained in 0.1 M Tris-HCl, pH 7.6 (mV = 718.9 - 41.1 log [S-l M), accurately describes the time course of the reaction for cysteine synthetase. The rate of volatility was determined for each assay and all points were corrected for loss of sulfide from the reaction mixture by volatilization. All time courses were recorded on paper tape using a Datos 308 analog to digital converter and clock interfaced to a Heath-Schlumberger Model EU 205-11 recorder. The time course was then linearized and plotted using a program, which includes all the features listed above, written for the Wang 720C programmable calculator with a flatbed plotter and an input/output writer. Time courses for cysteine synthase are shown in Fig. 1.
FIG. 1. Time courses for cysteine synthetase. Standard assay conditions were used. Cysteine synthetase concentrations are: (0) 0.071 mg/ml; (0) 0.142 mg/ml; (0) 0.284 mg/ml.
WEDDING As can be seen, the time courses exhibit a prominent lag prior to attaining the steady state. Initial velocities were calculated by performing a leastsquares fit to a linear regression equation on the linear portion of the time courses (indicated by arrows in Fig. 1). A plot of velocity, determined by the above method, versus enzyme concentration, in milligrams per milliliter, is linear. Data processilzg. Initial velocities were plotted against substrate concentrations, as well as the reciprocals of both. All calculations were carried out using programs written for the Wang 720C programmable calculator. The saturation curve for 0-acetylserine sulfhydrylase was fitted to the following equation by the method of Cleland (4): v = V A/(K,
+ A).
PI
The saturation curve for sulfide was fitted to the equation for substrate inhibition (assuming substrate binds to enzyme in two places) by a leastsquares method to a multiple linear regression equation: VA ’ = K, + (AZ/Ku)
+ A ’
All T’S were determined according to the method of Dixon and Webb (5). Analytical disc gel electrophoresis. Analytical polyacrylamide disc gel electrophoresis was carried out at room temperature with a Polyanalyst (Buchler Instruments Division) electrophoresis apparatus and a voltage and current regulated dc power supply, Model 3-1014A. All gels were run at a constant current of 3 n-&tube for approximately 2 h. The buffer system used was that of Reisfeld and Small (61, with either 6 or 8% acrylamide and the omission of 8 M urea. Gels were run in duplicate, after which one was stained, for 2 h, and destained using the method of Weber and Osborn (7). The duplicate gel was sliced into 1.5-mm pieces using an Ames Model 3542 lateral gel slicer and the slices were added to 0.5 ml of 0.1 M Tris-HCl, pH 7.6 and allowed to stand for 24 h in order to elute protein from the gel slices. Fractions were then assayed for both serine transacetylase and 0-acetylserine sulfhydrylase activities as described above. Enzymes. Cysteine synthetase was obtained from S. typhimurium LT-2 as a byproduct of the purification ofO-acetylserine sullhydrylase. 0-Acetylserine sulfhydrylase was purified by the method of Becker et al. (8), and the complex, cysteine synthetase, was eluted in the void volume of the first Sephadex G-100 chromatography step. The complex was further purified only by ammonium sulfate fractionation from 35-45% saturation. Cysteine synthetase had a final specific activity of 0.5 units/mg and was estimated
KINETIC
CHARACTERIZATR
)N
at 32% purity by the criterion of disc gel electrophoresis. Serine transacetylase was resolved from all Oacetylserine sulfhydrylase activity as described by Kredich and Tomkins (1). Chemicals. AR grade ammonium persulfate, phosphoric acid, hydrochloric acid, sucrose, sodium sulfide, and glycerol were obtained from Mallinckrodt. Electrophoresis grade acrylamide, NJ’-methylene bisacrylamide, N,N,N’,N’-tetramethylethylenediamine and glycine were obtained from Eastman Kodak Company. Enzyme-grade Tris buffer and enzyme-grade ammonium sulfate were obtained from Schwarz/Mann. Grade I flavin mononucleotide and S-acetyl CoA, sodium salt were obtained from Sigma Chemical Company. A grade L-serine and Oacetyl-L-serine were obtained from Calbiochem.
OF
CYSTEINE
295
SYNTHETASE
DTNB was obtained pany, Inc. Sephadex from Pharmacia.
from G-200
Aldrich Chemical Comand G-100 were obtained
RESULTS
If L-serine is varied over the concentration range lo-” to 10-l M at constant sulfide concentrations of 0.2 and 2 mM acetyl CoA, the saturation curve shown in Fig. 2 is obtained. Substrate inhibition is observed above concentrations of 5 mM. Moreover, there are indications of discontinuities, which are particularly apparent in the double-reciprocal plot inset in Fig. 2. An approximate S ,,.5 for L-serine is 0.14 rnM.
FIG. 2. Dependence of cysteine synthetase activity on the concentration of L-serine. Standard assay conditions were used except that the concentration of L-serine was varied as indicated and 0.2 IU of cysteine synthetase was used. Inset: LineweaverBurk plot.
(Sulfide)
When acetyl CoA is varied over the concentration range lo-” to 2.0 x low3 M with sulfide held constant at 0.25 mM and Lserine held constant at 5 mM, a saturation curve similar to that observed for r.,-serine is obtained again with indications of discontinuities. An approximate S,,., of 0.23 mM for acetyl CoA is obtained. When sulfide is varied over the concentration range 7 x 1O-7 to 3 x 10m3 M with acetyl CoA held constant at 2 mM and Lserine held constant at 5 mM, the saturation cure shown in Fig. 3 is obtained. Substrate inhibition is observed above 3 x 10e4 M and the curve is identical to that obtained for free 0-acetylserine sulfhydrylase with sulfide as the variable substrate (3).
hM)
3. Dependence of cysteine synthetase activity on the concentration dard assay conditions were used except that sulfide was varied as indicated cysteine synthetase was used. Inset: Lineweaver-Burk plot. FIG.
of sulfide. Stanand 0.35 IU of
296
COOK
AND
Addition of 0-acetylserine sulfhydrylase to the cysteine synthetase complex increases the overall rate of the reaction. This is shown in Fig. 4. The lowest point indicates the rate obtained with only the sulfhydrylase present in the complex as it is isolated. This value is obtained by dissociating the multienzyme complex to its component enzymes by incubation with 1 mM 0-acetyl-L-serine (2). The number of units which produces half-maximal velocity is 17.21 -+ 1.63. Lag studies. In a reaction initiated by the addition of enzyme to a solution of its substrates, the delay in reaching a steady-state rate as is shown for cysteine synthetase in Fig. 1 may occur due to a slow buildup in concentration of an intermediate along the reaction path. Starting the reaction with a substrate or pair of substrates, it was found that of the various combinations of initiating the reaction with single substrates or substrate pairs providing preincubation with various substrates, only where L-serine and acetyl CoA were incubated together with cysteine synthetase was the lag eliminated. This finding supports the hypothesis, since incubation with both substrates for the first enzyme in the se-
(OASS)
WEDDING
quence of the reaction catalyzed by the complex, serine transacetylase, would produce a buildup of 0-acetyl-L-serine, the most important intermediate along the reaction path. If this hypothesis is correct, the lag should reflect the saturation curves for the substrates of the first enzyme in the sequence of the reaction catalyzed by the complex, serine transacetylase. A plot of the reciprocal lag against the concentration of L-serine is shown in Fig. 5. The plot of UT against acetyl CoA concentration also reflects the saturation curve for acetyl CoA. The substrate inhibition observed for L-serine is also noted in the 7 plots. A plot of 7 against the reciprocal of sulfide concentration is shown in Fig. 6. As sulfide ion concentration increases, the lag is constant up to about 0.05 mM, after which the lag increases with increasing sulfide concentration. The fact that the lag remains constant prior to the increase at high sulfide confirms the assignment of a ping-pong mechanism to Oacetylserine sulfhydrylase (3) previously, since a ping-pong mechanism K, is decreased as the second substrate (sulfide) is decreased and, therefore, steady state is reached sooner. It has been determined (3) that sulfide is a substrate inhibitor of 0-acetylserine sulfhydrylase with a KIs2of 0.013 -+ 0.006 mM. The results obtained
(KU )
FIG. 4. Dependence of cysteine synthetase activity on exogenous 0-acetylserine sullhydrylase. Standard assay conditions were used except that Oacetylserine sulfhydrylase was added as indicated and 0.35 IU of cysteine synthetase was used. The lowest 0-acetylserine sulfhydrylase concentration is a measure of endogenous enzyme (4.5 IU) determined as indicated under Besults. Inset: Lineweaver-Burk plot.
(Serene)
FIG. 5. Dependence
(mM)
of reciprocal lag (l/7) for cysteine synthetase on L-serine concentration. Lags were obtained from the same assays used in the saturation curve for L-serine. Inset: Double-reciprocal plot.
KINETIC
CHARACTERIZATION
here are in reasonable agreement with the previously determined KI value. Therefore, sulfide only apparently increases the lag time through substrate inhibition of the sulfhydrylase. Stimulation of the rate of cysteine synthetase by addition of exogenous O-acetylserine sulfhydrylase is explained by considering that addition of this enzyme supplies more sites for binding of the intermediate 0-acetyl-L-serine. Therefore, the rate for the complex should increase until the number of sites available for the 0-acetyl-L-serine produced is saturating. The stimulation is not due to the fact that all the serine transacetylase is not complexed and more complex is formed as sulfhydrylase is added. This is shown below in the disc gel electrophoresis experiments and the Sephadex G-200 experiments which indicate a contamination of the complex by free 0-acetylserine sulfhydrylase. In this respect, O-acetylserine sulfhydrylase acts as a coupling enzyme. Assay for the intermediate, serine. If the intermediate,
0-acetyl-G
O-acetyl-nserine, is not transferred directly to the 0-acetylserine sulfhydrylase, then it must build up to a concentration suficient to allow it to bind to the O-acetylserine sulfhydrylase and the intermediate should be detected when cysteine synthetase is incubated with L-serine and acetyl
-.
FIG. 6. Dependence of lag (7) for cysteine synthetase on the reciprocal of sulfide concentration. Lags were obtained from the same assays used in the saturation curve for sulfide.
OF
CYSTEINE
SYNTHETASE
297
CoA alone. If, after the appropriate incubation, a large excess of 0-acetylserine sulfhydrylase is added followed by sulfide, the initial rapid disappearance of sulfide is a measure of 0-acetyl-L-serine. If a similar incubation is followed by the addition of DTNB, CoA can also be measured through its reaction with DTNB. The results of experiments of both these types are shown in Table I. A buildup of both products of serine transacetylase is indicated. This could be due to either the dissociation of the enzymes of the complex under the conditions of the above experiments or to the fact that the intermediate products, Oacetyl-L-serine and CoA, do not remain enzyme bound but rather dissociate from the transacetylase after formation. Dissociation assays. In order to determine whether or not cysteine synthetase does dissociate, enzyme samples incubated as above with and without substrates for the same approximate time period, were applied to a small Sephadex G200 column (1.4 cm x 0.4 cm21 and fractions eluted from the column were assayed for serine transacetylase using the DTNB assay and 0-acetylserine sulfhydrylase using the sulfide electrode. The control, Fig. 7A, in which 0.35 IU of cysteine synthetase was incubated in 0.1 M Tris-HCl, pH 7.6 for 4 min, shows coincidence of a portion of the sulfhydrylase activity with the transacetylase activity. This suggests that in addition to cysteine synthetase, free Odacetylserine sulfhydrylase is also present, since it has been shown previously (2) that complex formation is much favored. In the presence of saturating substrates for serine transacetylase, i.e., 2 mM acetyl CoA and 5 mM L-serine, the sulfbydrylase and transacetylase activities are resolved, as predicted by Kredich et al. (2); see Fig. 7B. If substrate levels are decreased to 0.6 mM L-serine and 0.4 mM acetyl CoA, there remains only a small amount of sulfhydrylase eluting with the transacetylase (Fig. 7D), again as predicted (2). These authors also suggested that sulfide prevents the dissociation of cysteine
298
COOK AND WEDDING TABLE
DETECTION
OF THE INTERMEDIATES
A Conditions
CoA CoA
0.2520 0.1852
B Conditions
0-ACETYL-L-SERINE,
A[~;$-lel
A[Sulfidel bud
m [FAZ
0.372 0.672
0.300
A
m ‘FAT
0.2520
0.0668
AOh
AND COENZYME
8-min incubation
0.1183
2-min incubation OD,,,
-L-Serine +L-Serine
I
SYNTHETASE,
4-min incubation
[S~llll~l -Acetyl +Acetyl
FOR CYSTEINE
0.1337
0.1337
4-min incubation m Y”Z 0.022
OD,,,
AOD,,,
0.370 0.970
0.600
‘(%y 0.044
Notes: (A) Incubations were carried out in the water-jacketed cell constructed for the sulfide ion selective electrode in a final volume of 1 ml with final concentrations of: L-serine, 5 mM; acetyl CoA, 2 mM; Tris-HCl, pH 7.6, 0.1 M; cysteine synthetase, 0.0095 IU. Acetyl CoA was omitted in the control. Excess 0-acetylserine sulfhydrylase (136 IU) was added prior to addition to sulfide. Sulfide concentration was recorded as in Methods. (B) Incubations were carried out in cells of l-cm light path with a final volume of 1 ml and final concentrations of: L-serine, 5 mM; acetyl CoA, 2 mM; DTNB, 1 mM; Tris-HCl, pH 7.6, 0.1 M; cysteine synthetase, 0.008 IU. Gserine was omitted from the control. Absorbance changes at 412 nm were recorded using a Gilford Model 2400 spectrophotometer. synthetase by 0-acetyl+serine. Under the same conditions as Fig. 7B, except that 0.5 mM sulfate was present, the results shown in Fig. 7C are obtained. Sulfide does prevent the dissociation of cysteine synthetase. In all cases, except the control, the large 280-nm peak eluting last contains the products of serine transacetylase, particularly CoA. If cysteine synthetase is incubated with 0.04 mM acetyl CoA and 0.04 mM L-serine, which would produce as much O-acetyl-Lserine as was measured in the experiments reported in Table I, the results shown in Fig. 7E are obtained. This figure produces qualitatively the same pattern as the control and the plus sulfide experiments (Figs. 7A and 7C), which indicates that cysteine synthetase does not dissociate under normal assay conditions. Disc gel experiments. Small amounts of free serine transacetylase could not be detected in the above experiments, since it would elute very near the complex peak. In order to determine if there was any dissociation, the same amount of enzyme which had been applied to the Sephadex G-200 columns were applied to 6 and 8% polyacerylamide gels. After electro-
phoresis, gels were sliced, protein was eluted, and fractions were assayed for both serine transacetylase and O-acetylserine sulfhydrylase activities. Results are shown in Fig. 8. Note that three bands of cysteine synthetase activity are observed. These three bands are probably aggregates as reported previously by ultracentrifuge studies (2). There is no detectable free serine transacetylase activity, but there are several bands of free 0-acetylserine sulfhydrylase activity. The last band migrates with purified 0-acetylserine sulfhydrylase and the other two bands migrate similarly to two contaminants reported by Becker et al. (8), who could detect no activity in these contaminants. However, this may have been a function of the fixed time calorimetric assay used in these studies. These bands may be aggregates of the sulfhydrylase. Serine transacetylase resolved from Oacetylserine sulfhydrylase, as in Methods, is active after disc gel electrophoresis. So, it appears that all serine transacetylase in the experiments presented is either part of the complex, cysteine synthetase, or was too low in concentration to
KINETIC
CHARACTERIZATION
,056.
OF
CYSTEINE
SYNTHETASE
299
1
026 _
-.12. E : ,-.
-
,”
-._ -z
T0.’ 056.
2.12. g. 028 L 0 2 v1 - z : -E t .048. > 072.
,024.
.I036.
D48.
124. -
C,
2
4
6
Fraction
Number
FIG. 7. Elution profiles from the Sephadex G-200 column used to determine dissociation of cysteine synthetase. The column was equilibrated with 0.1 M Tris-HCl, pH 7.6. Cysteine synthetase, 0.35 IU, was applied to the columns after different conditions of incubation. Total sample applied was 0.5 ml. (A), Cysteine synthetase incubated in 0.1 M Tris-HCl, pH 7.6; (B) cysteine synthetase incubated as in A with 5 mM L-serine and 2 mM acetyl CoA; (C) same conditions as B except that 0.25 mM sulfide was added; (D) cysteine synthetase incubated as in A with 0.6 InM L-serine and 0.4 mM acetyl CoA; (E) same conditions as in D except that 0.04 mM L-serine and 0.04 mM acetyl CoA were used. Serine transacetylase (O), 0-acetylserine sulfhydrylase (0), absorbance at 280 nm (0).
be detected by the assay used. When the experiment in Fig. 7E is repeated using disc gel electrophoresis, results identical to those presented in Fig. 8 are obtained. Additional kinetic data. If cysteine synthetase does release the intermediates, Oacetyl-L-serine and CoA, then if the first enzyme in the multienzyme complex is monitored via the DTNB assay, saturation curves identical to those obtained with the sulfide electrode should be observed. This serves two purposes: First, it supports the fact that the intermediates are released; second, since the DTNB assay is more sensitive than the electrode, more points can be obtained for both the
L-serine and acetyl CoA saturation curves. The saturation curve for L-serine is shown in Fig. 9. Note that two intermediate plateaus are observed in the curve and that they occur in the same substrate range as the apparent discontinuities detected with the electrode. The same kind of curve is observed for acetyl CoA as shown in Fig. 10. DISCUSSION
The saturation curve for L-serine obtained using both the sulfide electrode (Fig. 2) and the DTNB spectrophotometric assay (Fig. 9) displays two intermediary
300
COOK
w,ce
AND
WEDDING
N”rnbW
FIG. 8. A cysteine synthetase solution containing 0.71 mg of protein was applied to a 6% polyacrylamide gel. Slices (1.5 mm) of enzyme were extracted and assayed according to standard conditions, except that 0-acetylserine sullhydrylase was assayed using 0.044 mM sulfide. Samples were applied in 25% glycerol at a final volume of 0.1 ml. Three aggregates of cysteine synthetase are numbered in order of increasing molecular weight. Serine transacetylase (01, 0-acetylserine sulfhydrylase (0). I
/
0-acetylserine sulfhydrylase results in a stimulation of the rate of cysteine synthetase and does this because it acts in the capacity of a coupling enzyme and not because more complex is formed with free serine transacetylase since all of this enzyme has been shown to exist already in complex. The sulfhydrylase could only act in this capacity if the multienzyme complex cysteine synthetase released its Oacetyl-L-serine, as suggested. Data obtained from studying the lag as a function of substrate concentration leads to the interpretation that the lag is due to the need for buildup of enough O-acetyl-Lserine, the product of serine transacetylase, the first enzyme in the complex, to bind to 0-acetylserine sulfhydrylase, the first enzyme in the complex. The saturation curves for both substrates of serine transacetylase, which produces O-acetyl-Lserine, are reflected in plots of l/r against substrate concentration for the complex. Sulfide, however, since it has the ability to decrease apparent K, for O-acetyl-L-serine, has no effect on the lag until its concentration becomes high enough to be substrate inhibiting for 0-acetylserine sulfhydrylase. The above interpretation is confirmed by the fact that cysteine synthetase incubated with substrates for serine transacetylase produces large amounts of both Oacetyl+serine and CoA. This production
FIG. 9. Dependence of serine transacetylase activity in cysteine synthetase on the concentration of acetyl CoA. Standard assay conditions were used and all assays were initiated by addition of 0.02 IU of cysteine synthetase.
plateaus. An explanation of intermediary plateaus has been presented by Teipel and Koshland (9) which suggests that these plateaus are a result of mixed positive and negative cooperativity. A characterization of this phenomenon of cysteine synthase will require more investigation. Sulfide appears to produce a curve identical to that obtained for free O-acetylserine sulfhydrylase. This suggests there is no major alteration in the mechanism for the sulfhydrylase. Addition of exogenous
FIG. 10. Dependence of serine transacetvlase activity in cysteine synthetase on the concentration of L-serine. Standard assay conditions were used and all assays were initiated by addition of 0.006 IU of cysteine synthetase.
KINETIC
CHARACTERIZATION
of 0-acetyl-L-serine is not due to dissociation of cysteine synthetase under these conditions, as shown by both the Sephadex G-200 dissociation data and disc gel electrophoresis. Other multienzyme complexes which do not exhibit covalent enzyme-intermediate complexes have behavior which can be classified according to either release or nonrelease of intermediates. Several examples are found of the former class. Some are the multienzyme complex from Neurospora crassa (10) responsible for the biosynthesis of aromatic amino acids. In this complex, dehydroquinic acid is released from the enzyme dehydroquinic acid synthase if there is a block after this enzyme. The complex responsible for synthesis of isoleucine and valine in S. typhimurium (11) and N. crassa (12, 13) also releases the intermediate product. Tryptophan synthase from Escherichia coli (14) is an example of the latter clas. Even if there is a block after the protein of this complex, indole release is very slow. The measurement of O-acetyl-L-serine in Table I after incubation for 8 min produces enough intermediate to cause dissociation of the complex, yet the rate of production is still only twice that observed at 4 min. Therefore, it appears that cysteine synthetase, unlike most other complexes, allows release of its intermediate at the same rate whether in complex or free unless dissociation affected by 0-acetyl+serine is slow on the same time scale. It has been confirmed using chromatog raphy on Sephadex G-200 that O-acetyl-Lserine in excess of 10m4 M causes dissociation of the complex, cysteine synthetase, as shown by Kredich et al. (2>, and also, as these authors have indicated, that sulfide prevents this 0-acetyl-L-serine-dependent dissociation. Concentration-dependent aggregation of cysteine synthetase has also been reported (2) using the ultracentrifuge and has been noted in these studies using disc gel electrophoresis. Possible aggregation of O-acetylserine sulfhydrylase, which has not been reported previously, has been noted using the electrophoresis technique described. These probable aggregates may
OF
CYSTEINE
SYNTHETASE
301
have been observed by the above authors as two bands reported as contaminants running behind the main sullhydrylase band, but not detected due to the low sensitivity of the calorimetric assay. Lynen (15) has suggested several advantages of multienzyme complexes including minimization of competition with other pathways by keeping an intermediate in a limited microenvironment, increase in local concentration of the intermediate to the active site of the next enzyme in the sequence, regulatory features, protection of unstable intermediates, and creation of specific environments of different nature (e.g., hydrophobicity) to enhance specific reactions. Of these, the local concentrations increase and regulatory features seem to be the most likely for cysteine synthetase. Along with these possibilities, it may be considered that the multienzyme complex also decreases the lag in the overall reaction for both enzymes by decreasing the space which must be traversed by the intermediate. Easterby (16) has shown that in a coupling system, the lag for the overall reaction is equal to the simple sum of the K/V values for all enzymes after that which is rate limiting. By comparing the K/V value for 0-acetylserine sulfhydrylase against the limiting lag for the complex, i.e., in the absence of any free it can be determined sulfhydrylase, whether this latter possibility is of any significance. The K/V value for free sulfhydrylase is approximately 0.9 min, while for the same amount of sulfhydrylase in complex the lag is approximately 0.5 min. This indicates that the lag is decreased twofold by complex formation. There is also evidence for the regulatory aspects of the complex in the saturation curves for L-serine and acetyl CoA, and the possible aggregation phenomenon, but much work remains to be done in this area. Many other enzymes are known to undergo concentration-dependent polymerization. Some of these have been discussed by Nichol (17). It is somewhat more difficult to ascertain the significance of this phenomenon since cellular concentrations are not accurately known nor are local
302
COOK
AND
concentrations within the cell. One possibility is an increase in sensitivity to feedback inhibition, e.g., the feedback inhibitor induces association or dissociation changing activity. Since cysteine synthesis is feedback inhibited by L-cysteine at the level of serine transacetylase, there is a possibility for something of this nature to occur, but again more work must be done in this area. ACKNOWLEDGMENTS We wish to thank Dr. W. W. Cleland for his help with some of the data interpretation. We would also like to thank Kay Black for her help in proofreading and editing the manuscript. REFERENCES 1. KREDICH, N. M., AND TOMKINS, G. M. (1966) J. Biol. Chem. 241, 4955-4965. 2. KREDICH, N. M., BECKER, M. A., AND TOMKINS, G. M. (1969) J. Biol. Chem. 244,2428-2439. 3. COOK, P. F., AND WEDDING, R. T. (1976) J. Biol. Chem. 251, 2023-2029. 4. CLELAND W. W. (1967) Adv. Enzymol. 29, l-32.
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