ARCHIVES
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. l-7,
1991
Kinetic Characterization Dehydrogenase’ Bert Bayer’ Louisiana
Received
and Richard
State University
June
of Branched Chain Ketoacid
Odessey3
Medical
20, 1990, and in revised
Center, Department
form
October
of Physiology,
Inc.
70112-1393
to the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complex. All three enzymes (ketoacid dehydrogenases) reside in the mitochondrial matrix and play a regulatory role in intermediary metabolism ( 1). The ketoacid dehydrogenases are composed of three component enzymes which catalyze consecutive steps in the overall reaction. The dehydrogenase (El ) requires thiamine pyrophosphate (TPP) 4 as an essential cofactor, and catalyzes the ketoacid decarboxylation and formation of an hydroxyethyl-TPP intermediate ( Reaction [l] ) . Furthermore, El catalyzes the reductive acylation of the lipoic acid cofactor of the second component of the multienzyme complex, a dihydrolipoamide acyltransferase (E2, Reaction [ 21) . E2 transfers the acyl group to CoA (the acceptor) to produce the product (acyl CoA) of the overall reaction (Reaction [ 31) . Transfer of the acyl group leaves the lipoic acid moiety of E2 in the reduced state. However, lipoic acid is reoxidized by an FAD-containing dihydrolipoamide dehydrogenase (E3) which utilizes NAD+ as the final electron acceptor (Reactions [ 41 and [ 51) . The overall reaction [ 61 velocity was measured by determining the rate of NADH production. RCOCOzH + [ thiamine-PP] [ RCHOH-thiamine-PP] [ RCO-S-lipSH]
1 This work was supported by grants from the National Institutes of Health HL07098 (to B.B.) and GM32654 and GM37217 (to R.O.). ’ To whom correspondence should be addressed at The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609. FAX: (207) 288-5079. 3 Current address: Biosurface Technology, One Kendall Square, Cambridge, MA.
-El +
[ RCHOH-thiamine-PP] [ RCO-S-lipSH]
The branched chain complex catalyzes the oxidative decarboxylation of the transaminated products of the three branched chain amino acids, leucine, isoleucine, and valine. The branched chain complex is structurally similar
0003.9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
St., New Orleans, Louisiana
12, 1990
Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a pingpong mechanism and are similar to pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition. o 1991 Academic Press,
1901 Perdido
[lip(SH),]-E2
-El + CO2
[l]
-El + [ lipSZ] -E2 --* -E2 + [thiamine-PP]
-El
[2]
[ lip( SH),] -E2 + RCO-S-CoA
[3]
-E2 + CoA-SH + + [FAD]-E3
+
[lips,] -E2 + reduced [FAD]
-E3
[4]
* Abbreviations used: TPP, thiamine pyrophosphate; KIV, cu-ketoisovalerate; DTT, dithiothreitol; EGTA, ethylene glycol bis (@-aminoethyl ether)N,N-tetraacetic acid, PMSF, phenylmethylsulfonyl fluoride; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; PEG, polyethylene glycol. 1
Inc. reserved.
BOYER
2
AND
reduced [FAD] -E3 + NAD + + [FAD] -E3 + NADH + H+
[5]
Sum: RCOCOOH + CoA-SH + NAD+ + RCO-S-CoA + CO2 + NADH + H’
[6]
To understand the interaction of several feedback inhibitors, it was necessary to characterize the kinetic mechanism of the branched chain complex using initial velocity patterns and product inhibition studies. Initial velocity experiments were conducted by varying one substrate concentration at fixed concentrations of a second substrate, while keeping the remaining substrates at saturating concentrations. Initial velocity experiments only distinguish ping-pong from sequential reactions, while product inhibition experiments distinguish between mechanisms with different rate equation forms (2). Data collected from initial velocity and product inhibition experiments carried out with pyruvate dehydrogenase ( 3 ) and a-ketoglutarate dehydrogenase (4,5 ) are consistent with the patterns predicted from rate equations derived for a three-site ping-pong mechanism (6). To our knowledge, similar studies have not been performed using the branched chain complex. The kinetic constants were calculated for a three-site ping-pong mechanism derived by Cleland ( 7). EXPERIMENTAL
PROCEDURES
Materials. Female retired breeder rats ( Sprague-Dawley) weighing 250-600 g were obtained from Hilltop Laboratories (Scottsdale, PA) and were provided with Purina laboratory chow and water ad libidum. All biochemicals and enzymes were of the highest grade available, and almost all were obtained from Sigma. Benzyloxycarbonyl-Phe-Ala diazomethyl ketone ( Z-Phe-Ala) and benzyloxycarbonyl-Phe-Phe diazomethyl ketone (Z-Phe-Phe) were a generous gift from Dr. Elliott Shaw of the Friedrich Meischer-Institut (Basel, Switzerland). Hydroxylapatite ( Bio-Gel HTP) was obtained from Bio-Rad. Enzyme assays. All enzyme assays were performed at 37°C. cu-ketoisovalerate (KIV) (1 mM) was used as the ketoacid substrate in all experiments. Unless otherwise indicated, the assay buffer consisted of: 1 mM NAD+, 0.20 mM thiamine pyrophosphate, 0.061 unit/ml lipoic dehydrogenase, 0.1 mM coenzyme A, and 0.5 pg/ml rotenone. To assay the modulators of the branched chain complex, the assay buffer (1.0 ml total volume) was prepared with several concentrations of one cofactor, while adding fixed concentrations of competitive inhibitor just before the assay was to be performed. Following the addition of the competitive inhibitor, 0.025 units of purified branched chain complex were added with mixing, and then 20 ).d of KIV (50 mM) was added with mixing. The production of NADH was monitored fluorometrically for approximately 1 min to determine the initial rate of the reaction. All kinetic constants were fit to the nonlinear Michaelis-Menten equation for competitive inhibitors (7)) and implemented on either an IBM 3081 mainframe using the MVS/XA operating system with the SAS-nonlinear regression procedure, or a PCXT microcomputer using the Pennzyme nonlinear kinetics program generously supplied by Dr. David Garfinkel (8). Competitive inhibition was assumed if there was no statistically significant difference in the V, of individual curves at fixed concentrations of the other substrates. Purification of the branched chain complex. The following were used in the purification of the branched chain complex.
buffers
ODESSEY Buffer A: 20 mM Hepes, 0.5 mM MgClz, 1 mM DTT, and 1 mM benzamidine. Buffer B: 50 mM Hepes, 1 mM benzamidine, 6 mM MgClz, 5 mM EGTA, 1 mM DTT, 0.5 mM PMSF (dissolved in ethanol), and 0.05 mM chymostatin (dissolved in DMSO) . Buffer C: 2 mM trypsin inhibitor and 200 units/ml aprotinin in buffer A. Buffer D: 50 mM Hepes, 20% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM benzamidine, 1.0 mM DTT, 0.5 mM PMSF, 2.0 mM MgClz, and 0.05 mM chymostatin. All buffers were adjusted to pH 7.3 with 1 N KOH. Isolation buffer: 220 mM mannitol, 70 mM sucrose, 2 mM Hepes, and 0.5 mM bovine serum albumin (BSA, Fraction V) pH 7.4. Retired breeder rats were sacrificed by an Mitochondria isolation. ether overdose and kidneys were obtained within 5 min of death and placed in approximately 1 vol (w/v) of ice cold isolation buffer. The kidneys were ground with a meat grinder and suspended in 2 vol of isolation buffer. The pH of the slurry was adjusted to 7.4 with 1 N NaOH. The tissue was then homogenized in a continuous flow homogenizer (9) at 760 rpm and the pH was adjusted to 7.4 and immediately centrifuged in a Beckman JAlO rotor by allowing the rotor to attain a peak speed of 4500 rpm and stopped with the brake at 7. The supernatant was carefully aspirated off and saved. The remaining pellet was resuspended in 1 vol isolation buffer using the continuous flow homogenizer, the pH was readjusted to 7.4, and the initial spin was repeated. The resulting supernatant was combined with the initial supernatant and this mixture was centrifuged in a Beckman JAlO rotor at 16,000g for 23 min. The supernatant was discarded and the pellet was resuspended in a volume of isolation buffer approximately equal to 0.5 ml/g original tissue weight. Digitonin (20 mg/ml) was dissolved in isolation buffer by slowly heating on a hot plate stirrer until the solution was clear, then the digitonin was cooled on ice. Digitonin was added to the microsomal preparation (0.2 mg/mg protein) with stirring at 4°C. Three minutes after the addition of the digitonin, 3 vol of isolation buffer was added and the solution was centrifuged for 20 min at 16,000g. The supernatant was removed and the mitochondrial pellet was resuspended in buffer A with the continuous flow through homogenizer, and then centrifuged again at 16,000g for 20 min. This step was repeated, and the final pellet was resuspended in buffer A with a hand held Teflon homogenizer, to a final concentration of approximately 20-50 mg protein/ml. The mitochondria were then frozen in a large Erlenmeyer flask that was precooled by swirling it in liquid nitrogen. Once the slurry was frozen in liquid nitrogen, it was stored at -8O’C for up to 3 months with no loss in activity. The mitochondria were thawed by removing the Erlenmeyer from the -80°C freezer and warming the contents slowly at room temperature. Once the mitochondrial slurry began to thaw, protease inhibitors were added with constant swirling until completely thawed. The solution of inhibitors consisted of 0.8 vol of buffer B, 0.1 vol of buffer C, and 0.001 vol of (Z-Phe-Phe) and (Z-Phe-Ala) (10 mM in DMSO), and 0.5 mM PMSF. After the pellet was thawed, the solution was kept at 4°C throughout the remainder of the purification. The freeze-thaw solution was centrifuged at 20,400g for 30 min with the brake at MAX. This pelleted the mitochondrial inner membrane (which was discarded) and the matrix enzymes were released in the supernatant (freeze-thaw supernatant). With stirring, 50% polyethylene glycol (PEG 6000mw) was added to the freeze-thaw supernatant to a final concentration of 2% (v/v). After stirring at 4°C for 20 min, the mixture was centrifuged for 10 min at 20,400g. The PEG pellet was discarded, and additional PEG was added to the supernatant to a final concentration of 4%. After another 20.min stirring on ice, the centrifugation step was repeated and the 4% pellet was resuspended in buffer D using a motor driven Teflon homogenizer (760 rpm) to obtain a final concentration of approximately 3 units branched chain complex/ml. The solution was then stored at -8O”C, or applied to the hydroxylapatite column. The solution was clarified by centrifugation (19,OOOg, 10 min) before it was applied to the hydroxylapatite column.
BRANCHED I
I
I
I
CHAIN 1 *
COMPLEX
3
KINETICS
chain complex was consistent with a three-site ping-pong mechanism (6). In the absence of products, the rate equation for a three-site ping-pong reaction mechanism with two ping-pong sites (6) can be reduced to:
V Ka Kb l+[Al+[Bl+[Cl
ll=
I
I
II
30
I
I
50 70 KoAI-‘, (mu)-’
I
90
FIG. 1. Double reciprocal plot for CoA versus KIV. Initial velocity pattern when CoA was varied at fixed concentrations of added KIV of 0.50 (O), 0.125 (O), 0.042 (X), and 0.031 mM (B). NAD was held constant at 1 mM, TPP at 0.2 mM, and lipoamide concentration was 0.061 units/ml.
The clarified PEG supernatant (up to 15 mg protein) was layered on a hydroxylapatite column at a flow rate 0.25 ml/min (larger columns were used for larger protein preparations and the flow was increased accordingly so that the bed volume of the column could be washed every 30-45 min) . After all of the protein had entered the column, the protein was washed with degassed 140 mM potassium phosphate buffer containing 1 mM DTT and 0.2 mM TPP, pH 7.3, until the initial protein peak (which contained the pyruvate dehydrogenase complex) was eluted, and no more pyruvate dehydrogenase activity could be assayed (the pyruvate dehydrogenase assay was identical to the branched chain complex assay, except the ketoacid substrate was pyruvate) Following the complete elution of the pyruvate dehydrogenase complex a linear gradient of phosphate buffer was then applied to the column (140-560 mM phosphate, 1 mM DTT, and 0.2 mM TPP, pH 7.3) over a 2- to 4-h period and at a flow rate of 0.25-1.25 ml/min (depending on the size of the column) with a programmable Ultrograd gradient maker (LKB) and constant flow pump (Isco) . An optical density scanner recorded the absorbance of the eluant from the column and allowed us to identify the protein peaks. All protein fractions were collected and assayed for branched chain complex activity. Active fractions were collected (220-280 mM phosphate buffer), and 0.81 units lipoamide/ml active fractions were added to the combined fractions which were then centrifuged in a Beckman Ti 50 rotor at 165,300g for 2 h. The final pellet was resuspended in buffer D, to a concentration of approximately S-10 units/ml. Before freezing, the branched chain complex was clarified by centrifugation at 15,000g for 15 min. The enzyme was then stored in 50200 rl aliquots at -80°C for 2-3 months without any loss of activity. The specific activity of this preparation ranges from 3 to 4 U/mg and was homogeneous on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified branched chain complex samples were also analyzed for protein content (lo), to determine the specific activity of the branched chain complex. BSA was used as the protein standard in the assay. The nomenclature used throughout the kinetic analysis is that described by Cleland (7).
RESULTS
The first series of experiments was designed to determine whether or not the overall reaction of the branched
Kc ’
where K,, Kb, and Kc are the Michaelis constants for the ketoacid, CoA, and NAD+ , respectively (3 ) . Both the lipoyl dehydrogenase and the ketoacid decarboxylating sites are assumed to be ping-pong ( 6). Initial velocity patterns obtained when one substrate concentration (KIV or CoA) was varied in the presence of a series of fixed concentrations of a second substrate ( CoA or NAD+) resulted in double reciprocal plots with a series of parallel lines, consistent with a ping-pong mechanism (Figs. 1 and 2). Since these results were consistent with the ping-pong mechanism, it was not necessary to vary KIV at a series of fixed concentrations of NAD+. The kinetic constants for ketoisovalerate, ketoisocaproate, ketomethylvalerate, CoA, and NAD+ were 18.3 1- 2.1, 16.7 f 1.4, 10.5 + 1.5, 10.5 XL 2.0, and 49.3 f 6.5 PM, respectively. Inhibition of the reaction by product inhibitors of the branched chain complex were also investigated. In a threesite ping-pong mechanism, products binding at the same site the substrate binds were expected to exhibit competitive inhibition patterns. Furthermore, products binding to other substrate binding sites were expected to exhibit uncompetitive patterns, since the individual site reactions were ping-pong (Figs. 1 and 2). NADH and isovaleryl CoA inhibited branched chain complex activity competitively with respect to their sub-
,
I
I
I
I .
.36 -i
1
3
12 NAZI:-‘, (mM) ”
FIG. 2. Double reciprocal plot pattern when NAD was varied were 0.10 (0). 0.033 (O), 0.02 constant at 1 mM, TPP at 0.2 0.061 units/ml.
for NAD+ versus CoA. Initial velocity at fixed concentrations of added CoA (X), and0.011 mM (m). KIV was held mM, and lipoamide concentration was
4
BOYER
AND
ODESSEY
1
I I
I -2
I 1 4 MADl-B
!I 1
1 l2
I 16
(Id) -’ FIG. 3. Product inhibition NAD. The concentrations of (X), and 0.03 mM (m). KIV, mM, respectively. Lipoamide
by NADH at varying concentrations of added NADH were 0 (0)) 0.01 (O), 0.02 TPP, and CoA were held at 1, 0.2, and 1 concentration was 0.061 units/ml.
strate precursors (Figs. 3 and 4). In addition, isovaleryl CoA and NADH both inhibited the branched chain complex uncompetitively with respect to KIV (Figs. 5 and 6). However, NADH versus CoA (Fig. 7) and isovaleryl CoA versus NAD+ (Fig. 8) did not conform to the expected inhibition pattern. Inhibition of the branched chain complex by isovaleryl CoA versus CoA at nonsaturating NAD+ concentrations (0.125 and 0.060 mM) maintained a competitive inhibition pattern (data not shown). Secondary plots (slope replots) of slope versus isovaleryl CoA concentration were also linear. With decreasing NADf concentrations the Kzp for CoA, Kpp for isovaleryl CoA, and the V zp decreased (Table I), as would be expected with a ping-pong mechanism (2). However, the K”,pP/ V “,pp ratio decreases as
T
.36 -32 t -&- .28 E .24 1 -20 T - .16 i .12 .08 -04
2
I
I
1
40
20
lo
I I 50
[KM-I,‘irnM)-I FIG. 6. Product inhibition by isovaleryl CoA versus KIV. The concentrations of added isovaleryl CoA were 0 (0)) 0.05 (0) , and 0.10 mM ( X ) . NAD, TPP, and CoA were held at 2,0.2, and 0.05 mM, respectively. Lipoamide concentration was 0.061 units/ml.
the NAD+ concentration decreases and is inconsistent with a ping-pong mechanism [ ping-pong mechanism predicts no change in the Kzp/ V zp ratio, (2)]. The competitive inhibition pattern for NADH versus NAD’ at nonsaturating CoA concentrations (0.033 and 0.016 mM) was maintained. Unexpectedly, the slope replots were parabolic (Fig. 9). The K$P for NAD+, K?P for NADH and V $‘P decreased as the CoA concentration decreased (Table I). These observations cannot be explained by the ping-pong mechanism formulated for the other ketoacid dehydrogenase complexes (6). DISCUSSION
Although some kinetic properties of the branched chain complex have been elucidated (11-14)) this early work represents a collection of data obtained from partially purified enzyme preparations and several different cofactor concentrations were used. To our knowledge, a com-
I ’
1
I
I
I
I
f= I
I
-20
I lo
I
30
I
--I
90 wl
FIG. 4. Product inhibition of CoA. The concentrations (O), 0.02 (X), and 0.03 mM 0.2, and 1 mM, respectively. ml.
I
-?(mM)-~”
by isovaleryl CoA at varying concentrations of added isovaleryl CoA were 0 (0) , 0.01 (W). KIV, TPP, and NAD were held at 1, Lipoamide concentration was 0.061 units/
I
I
2
la
I
20
I
1
I
30
40
50
[KM-‘, (mu)-’ FIG. 6. Product inhibition by NADH versus KIV. The concentrations of added NADH were 0 (0)) 0.01 (O), and 0.03 mM (X ) . NAD, TPP, and CoA were held at 0.8, 0.2, and 0.05 mM, respectively. Lipoamide concentration was 0.061 units I ml.
BRANCHED
& 5
0.4
2 z
0.3
CHAIN
COMPLEX
5
KINETICS
0.1
FIG. 7. Product inhibition by NADH versus CoA. The concentrations of added NADH were 0 (0)) 0.0125 (0)) and 0.025 mM (X). NAD, TPP, and KIV were held at 1, 0.2, and 1 mM, respectively. Lipoamide concentration was 0.061 units/ml.
FIG. 8. Product inhibition by isovaleryl CoA versus NAD+. The concentrations of added isovaleryl CoA were 0 ( 0 ) , 0.01 (0 ) ,0.02 mM ( X ) , and 0.03 mM (m) . CoA, TPP, and KIV were held at 0.1,0.2, and 1 mM, respectively. Lipoamide concentration was 0.061 units/ml.
plete steady state kinetic analysis of the branched chain complex has not been performed prior to this investigation. Our experimental design was based on the work of Tsai et al. (3)) who performed the first steady state kinetic analysis of the pyruvate dehydrogenase complex using recently developed rate equations for a three-site pingpong mechanism (6). We have also correlated the branched chain complex initial velocity and product inhibition patterns with patterns predicted by the rate equations derived by Cleland for a three-site ping-pong mechanism. Inhibition by NADH versus NAD+ and isovaleryl COA versus CoA was competitive, while the inhibition by NADH or isovaleryl CoA versus KIV was uncompetitive, as predicted by the rate equations. These patterns were
similar to those reported for the pyruvate dehydrogenase (3) and a-ketoglutarate complex (4). Exceptions to the patterns predicted by the rate equations, present in all three ketoacid dehydrogenase complexes [ (3, 4) and present study], were the inhibition patterns observed for isovaleryl CoA versus NAD+ and NADH versus CoA, which were predicted to be uncompetitive. NADH versus CoA resembles a mixed type of inhibition with the lines intersecting in the third quadrant. The reciprocal plot of isovaleryl CoA versus NADf yields a set of lines which fail to converge in a single quadrant. These results could not be compared to the data of Tsai et al. (3) or Hamada et al. (4)) since they only used one inhibitor concentration. Cleland (6) has attempted to explain the unexpected inhibition patterns obtained by Tsai et al. ( 3 ) . Noncom-
TABLE Kinetic Competitive inhibitor Isovaleryl-CoA
NADH
Fixed [substrate] NAD (1 mM) (0.125 mM) (0.060 mM) CoA (1 mM) (0.1 mM) (0.033 mM) (0.017 mM)
Constants
for
Nonsaturating
I Product
Inhibition
Varied substrate KfP CoA 7.4 f 1.0 3.7 k 0.7 1.6 + 0.4 NAD 46.9 rfr 49.3 + 42.7 f 34.0 f
5.3 6.5 5.0 6.0
Slope replots
10.5 f 2.0 2.5 f 0.4 1.1 f 0.3
10 + 0.3 7.5 f 0.2 4.2 k 0.1
0.75 0.49 0.38
1.42 0.68 0.69
Linear -
10.2 12.7 9.7 6.7
14.4 16.3 11.9 11.1
3.26 3.02 3.59 3.12
0.22 0.26 0.23 0.20
Parabolic
* + f +
1.5 2.5 1.0 1.0
f f f +
0.3 0.5 0.3 0.4
Note. Product inhibition experiments were carried out as described in the methods and illustrated in Figs. 3 and 4. The Kzp, Kz!‘, and Vz*, are apparent Michaelis constants (K, app for the varied substrate, KT” for the inhibitor, and Vzp for the velocity expressed in nmol NADH formed per minute). All Michaelis constants were calculated using the nonlinear regression as described in the methods. The KIV and TPP concentration was held constant at 1 mM and the lipoamide concentration The isovaleryl CoA and NADH concentration ranged from 0.005 to 0.3 mM. All constants are listed as means f S.D. and are
-
abbreviations used, apparent maximal program Pennzyme was 0.061 unit/ml. in (PM).
6
BOYER
AND
7 5 0
4
8
12
16
20
24
28
FIG. 9. Secondary plot of the slope versus inhibitor concentration for inhibition by NADH at fixed CoA concentrations. The CoA concentrations were 0.1 (+ ) , 0.033 (O), and 0.016 mM (Cl). KIV was held constant at 1 mM, TPP at 0.2 mM, and lipoamide concentration was 0.061 units/ml.
petitive inhibition patterns were observed for acetyl CoA versus NAD+ and for NADH versus CoA in the pyruvate dehydrogenase complex (3). He concludes that a product will be noncompetitive against the substrate that binds at the next site in the overall reaction sequence if that site is random sequential. In addition, if a product combines as a dead-end inhibitor at another site, or hinders the reaction at this other site, it will be noncompetitive versus the substrate that normally binds at that site (3). In contrast to the observations of Tsai et al. (3) and Hamada et al. (4)) we observed a mixed type of inhibition for NADH versus CoA (Fig. 7) and a set of lines which fail to converge in a single quadrant for isovaleryl CoA versus NAD+ (Fig. 8). To further explain these anomalous patterns Tsai et al. (3) investigated the effect of pyruvate and CoA on theflavoprotein-catalyzedreaction (lipoamide + NADH + H+ + dihydrolipoamide + NAD+) . CoA, but not pyruvate, inhibited the complexed flavoprotein (El-E2E3) noncompetitively with respect to NADH and lipoamide ( 3 ) . In addition, noncompetitive inhibition patterns were observed with CoA versus NAD+ and CoA versus dihydrolipoamide ( reverse reaction), while CoA and pyruvate failed to inhibit the uncomplexed protein. Two explanations were proposed on the basis of these findings ( 3 ) : (i) The first possibility is that the transacetylase (E2-core) and flavoprotein (E3) catalytic centers are so close that binding of CoA (or acyl CoA) to E2 sterically hinders binding of NADH (or NAD+) to E3, and visa versa. (ii) The second possibility is that the binding of CoA (or acyl CoA) to E2 causes a conformational change in E2 that hinders binding of NADH (or NAD+) to E3.
ODESSEY
It appears that all three intramitochondrial multienzyme complexes have similar kinetic E2-E3 interactions. This is not surprising since the E3 component of all three complexes is thought to be identical ( 1). We attempted to describe the interaction of two potential physiological feedback inhibitors (NADH and isovaleryl CoA) by studying their inhibition patterns at nonsaturating concentrations of “fixed” substrate (the fixed substrate binds at a different site than the substrate being varied). Rate equations for a ping-pong mechanism state that the ratio of Kzp/ V “,pp is independent of the fixed substrate concentration and, therefore, should not change as the concentration of fixed substrate is reduced (2). A fixed KzP/VzP ratio is predicted for a ping-pong reaction because the apparent Michaelis constants increase to their limiting values as the fixed substrate concentration increases, and decrease as the fixed substrate concentration decreases. For a sequential reaction however, the Michaelis constants may change in any direction as the fixed substrate concentration increases, since the change in Michaelis constant depends on whether the Ki, is less than, greater than, or equal to the K, (2 ) . The apparent V,,, is the same for both types of reactions. Therefore, as the fixed substrate concentration increases, the K”,pp for a sequential reaction must decrease or remain constant (2). As seen in Table I, the K$P for CoA increases as NAD+ (fixed substrate) increases and the Kzp for NAD+ increases as the CoA concentration increases. This suggests that both reactions are indeed pingpong. The fixed substrates were varied from nonsaturating to saturating concentrations (Table I). For the inhibition of the branched chain complex by NADH, the K$‘P/VampP ratio does not change (Table I). Conversely, the Kzp/ V “,ppratio for inhibition by isovaleryl CoA at fixed NAD+ concentrations does tend to decrease. This observation does not confirm a sequential or ping-pong step in catalysis (2). We also observed a increase in the K!PP/ Kzp ratio (isovaleryl CoA/CoA) with increases in the level of fixed second substrate (NAD+) . In contrast to these results, Smith et al. ( 5) performed similar product inhibition studies with a-ketoglutarate dehydrogenase and found that as NAD” levels were increased, the KqPP/ Kzp for succinyl CoA versus CoA remained constant. Although the slope replots for the inhibition of the branched chain complex by isovaleryl versus CoA were linear, replots obtained for NADH versus NAD+ were parabolic. If an inhibitor binds at two different mutually exclusive sites, a linear mixed type of inhibition can occur and the system resembles a mixture of pure competitive and pure noncompetitive inhibition ( 15). This type of inhibition results in a failure of the reciprocal plots to converge at a common point and slope replots are parabolic. NADH has consistently been observed to be a pure competitive inhibitor against NAD; however, binding of NADH to an additional noncompetitive
BRANCHED
CHAIN
site has not been reported to our knowledge. It is therefore possible that a linear mixed type of inhibition does not account for the observed inhibition pattern. Further experiments are required to define this complex inhibitory pattern. In conclusion, the data we have obtained on the steady state kinetics of the branched chain complex correspond well to a three-site ping-pong mechanism (6). The exceptions include the inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA. Interestingly, we have observed a linear mixed type of inhibition pattern for NADH versus NAD + at nonsaturating CoA concentrations. To our knowledge, this has not been reported for the pyruvate dehydrogenase or a-ketoglutarate dehydrogenase complexes. Although subtle differences in the catalytic mechanism of the branched chain complex, pyruvate dehydrogenase complex, and ol-ketoglutarate dehydrogenase complex exist, these three multienzyme complexes appear to fit a three-site ping-pong mechanism. It is clear that further work is necessary to more precisely define their catalytic mechanisms. This kinetic study of the branched chain complex affords a better understanding toward this goal.
COMPLEX
KINETICS
7
REFERENCES 1. Yeaman, S. J. (1986) Trends in Biochem. Sci. 11,293-296. 2. Cleland, W. W. (1963) Biochem. Biophys. Acta 67,104-137. 3. Tsai, C. S., Burgett, M. W., and Reed, L. J. (1973) J. Biol. Chem. 248,8348-8352. 4. Hamada, M., Koike, K., Nakaula, Y., Hiraoka, T., Koike, M., and Hashimoto, T. (1975) J. Biochem. 77, 1047-1056. 5. Smith, C. M., Bryla, J., and Williamson, J. R. ( 1974) J. Biol. Chem. 249,1497-1505. 6. Cleland, W. W. (1973) J. Biol. Chem. 248,8353-8355. 7. Cleland, W. W. ( 1970) in The Enzymes (Boyer, P. D., Ed.), 3rd ed., Vol. 2, pp. l-65, Academic, New York. L. E., and Garfinkel, D. (1979) Camp. Biamed. 8. Kohn, M. C., Menten, Res. 12,461-469. 5, 2932-2938. 9. Ziegler, D. M., and Pettit, F. H. (1966) Biochemistry 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 10. Lowry, (1951) J. Biol. Chem. 193,265-275. 11. Parker, P. J., and Randle, P. J. (1978a) FEBS Z&t. 90, 183-186. 12. Parker, P. J., and Randle, P. J. (1978b) Biochem. J. 171,751-757. 13. Danner, D. J., Lemmon, S. K., Besharse, J. C., and Elsas, L. J. (1979) J. Biol. Chem. 254,5522-5526. S. J., and Reed, L. J. ( 1978) Proc. Natl. Acad. 14. Pettit, F. H., Yeaman, Sci. USA 75,4881-4885. in Enzyme Kinetics: Behavior and Analysis of 15. Segel, I. H. (1975) Rapid Equilibrium and Steady-State Enzyme Systems, pp. 176178, Wiley, New York.
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. l-7,
1991
Kinetic Characterization Dehydrogenase’ Bert Bayer’ Louisiana
Received
and Richard
State University
June
of Branched Chain Ketoacid
Odessey3
Medical
20, 1990, and in revised
Center, Department
form
October
of Physiology,
Inc.
70112-1393
to the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complex. All three enzymes (ketoacid dehydrogenases) reside in the mitochondrial matrix and play a regulatory role in intermediary metabolism ( 1). The ketoacid dehydrogenases are composed of three component enzymes which catalyze consecutive steps in the overall reaction. The dehydrogenase (El ) requires thiamine pyrophosphate (TPP) 4 as an essential cofactor, and catalyzes the ketoacid decarboxylation and formation of an hydroxyethyl-TPP intermediate ( Reaction [l] ) . Furthermore, El catalyzes the reductive acylation of the lipoic acid cofactor of the second component of the multienzyme complex, a dihydrolipoamide acyltransferase (E2, Reaction [ 21) . E2 transfers the acyl group to CoA (the acceptor) to produce the product (acyl CoA) of the overall reaction (Reaction [ 31) . Transfer of the acyl group leaves the lipoic acid moiety of E2 in the reduced state. However, lipoic acid is reoxidized by an FAD-containing dihydrolipoamide dehydrogenase (E3) which utilizes NAD+ as the final electron acceptor (Reactions [ 41 and [ 51) . The overall reaction [ 61 velocity was measured by determining the rate of NADH production. RCOCOzH + [ thiamine-PP] [ RCHOH-thiamine-PP] [ RCO-S-lipSH]
1 This work was supported by grants from the National Institutes of Health HL07098 (to B.B.) and GM32654 and GM37217 (to R.O.). ’ To whom correspondence should be addressed at The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609. FAX: (207) 288-5079. 3 Current address: Biosurface Technology, One Kendall Square, Cambridge, MA.
-El +
[ RCHOH-thiamine-PP] [ RCO-S-lipSH]
The branched chain complex catalyzes the oxidative decarboxylation of the transaminated products of the three branched chain amino acids, leucine, isoleucine, and valine. The branched chain complex is structurally similar
0003.9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
St., New Orleans, Louisiana
12, 1990
Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a pingpong mechanism and are similar to pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition. o 1991 Academic Press,
1901 Perdido
[lip(SH),]-E2
-El + CO2
[l]
-El + [ lipSZ] -E2 --* -E2 + [thiamine-PP]
-El
[2]
[ lip( SH),] -E2 + RCO-S-CoA
[3]
-E2 + CoA-SH + + [FAD]-E3
+
[lips,] -E2 + reduced [FAD]
-E3
[4]
* Abbreviations used: TPP, thiamine pyrophosphate; KIV, cu-ketoisovalerate; DTT, dithiothreitol; EGTA, ethylene glycol bis (@-aminoethyl ether)N,N-tetraacetic acid, PMSF, phenylmethylsulfonyl fluoride; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; PEG, polyethylene glycol. 1
Inc. reserved.
BOYER
2
AND
reduced [FAD] -E3 + NAD + + [FAD] -E3 + NADH + H+
[5]
Sum: RCOCOOH + CoA-SH + NAD+ + RCO-S-CoA + CO2 + NADH + H’
[6]
To understand the interaction of several feedback inhibitors, it was necessary to characterize the kinetic mechanism of the branched chain complex using initial velocity patterns and product inhibition studies. Initial velocity experiments were conducted by varying one substrate concentration at fixed concentrations of a second substrate, while keeping the remaining substrates at saturating concentrations. Initial velocity experiments only distinguish ping-pong from sequential reactions, while product inhibition experiments distinguish between mechanisms with different rate equation forms (2). Data collected from initial velocity and product inhibition experiments carried out with pyruvate dehydrogenase ( 3 ) and a-ketoglutarate dehydrogenase (4,5 ) are consistent with the patterns predicted from rate equations derived for a three-site ping-pong mechanism (6). To our knowledge, similar studies have not been performed using the branched chain complex. The kinetic constants were calculated for a three-site ping-pong mechanism derived by Cleland ( 7). EXPERIMENTAL
PROCEDURES
Materials. Female retired breeder rats ( Sprague-Dawley) weighing 250-600 g were obtained from Hilltop Laboratories (Scottsdale, PA) and were provided with Purina laboratory chow and water ad libidum. All biochemicals and enzymes were of the highest grade available, and almost all were obtained from Sigma. Benzyloxycarbonyl-Phe-Ala diazomethyl ketone ( Z-Phe-Ala) and benzyloxycarbonyl-Phe-Phe diazomethyl ketone (Z-Phe-Phe) were a generous gift from Dr. Elliott Shaw of the Friedrich Meischer-Institut (Basel, Switzerland). Hydroxylapatite ( Bio-Gel HTP) was obtained from Bio-Rad. Enzyme assays. All enzyme assays were performed at 37°C. cu-ketoisovalerate (KIV) (1 mM) was used as the ketoacid substrate in all experiments. Unless otherwise indicated, the assay buffer consisted of: 1 mM NAD+, 0.20 mM thiamine pyrophosphate, 0.061 unit/ml lipoic dehydrogenase, 0.1 mM coenzyme A, and 0.5 pg/ml rotenone. To assay the modulators of the branched chain complex, the assay buffer (1.0 ml total volume) was prepared with several concentrations of one cofactor, while adding fixed concentrations of competitive inhibitor just before the assay was to be performed. Following the addition of the competitive inhibitor, 0.025 units of purified branched chain complex were added with mixing, and then 20 ).d of KIV (50 mM) was added with mixing. The production of NADH was monitored fluorometrically for approximately 1 min to determine the initial rate of the reaction. All kinetic constants were fit to the nonlinear Michaelis-Menten equation for competitive inhibitors (7)) and implemented on either an IBM 3081 mainframe using the MVS/XA operating system with the SAS-nonlinear regression procedure, or a PCXT microcomputer using the Pennzyme nonlinear kinetics program generously supplied by Dr. David Garfinkel (8). Competitive inhibition was assumed if there was no statistically significant difference in the V, of individual curves at fixed concentrations of the other substrates. Purification of the branched chain complex. The following were used in the purification of the branched chain complex.
buffers
ODESSEY Buffer A: 20 mM Hepes, 0.5 mM MgClz, 1 mM DTT, and 1 mM benzamidine. Buffer B: 50 mM Hepes, 1 mM benzamidine, 6 mM MgClz, 5 mM EGTA, 1 mM DTT, 0.5 mM PMSF (dissolved in ethanol), and 0.05 mM chymostatin (dissolved in DMSO) . Buffer C: 2 mM trypsin inhibitor and 200 units/ml aprotinin in buffer A. Buffer D: 50 mM Hepes, 20% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM benzamidine, 1.0 mM DTT, 0.5 mM PMSF, 2.0 mM MgClz, and 0.05 mM chymostatin. All buffers were adjusted to pH 7.3 with 1 N KOH. Isolation buffer: 220 mM mannitol, 70 mM sucrose, 2 mM Hepes, and 0.5 mM bovine serum albumin (BSA, Fraction V) pH 7.4. Retired breeder rats were sacrificed by an Mitochondria isolation. ether overdose and kidneys were obtained within 5 min of death and placed in approximately 1 vol (w/v) of ice cold isolation buffer. The kidneys were ground with a meat grinder and suspended in 2 vol of isolation buffer. The pH of the slurry was adjusted to 7.4 with 1 N NaOH. The tissue was then homogenized in a continuous flow homogenizer (9) at 760 rpm and the pH was adjusted to 7.4 and immediately centrifuged in a Beckman JAlO rotor by allowing the rotor to attain a peak speed of 4500 rpm and stopped with the brake at 7. The supernatant was carefully aspirated off and saved. The remaining pellet was resuspended in 1 vol isolation buffer using the continuous flow homogenizer, the pH was readjusted to 7.4, and the initial spin was repeated. The resulting supernatant was combined with the initial supernatant and this mixture was centrifuged in a Beckman JAlO rotor at 16,000g for 23 min. The supernatant was discarded and the pellet was resuspended in a volume of isolation buffer approximately equal to 0.5 ml/g original tissue weight. Digitonin (20 mg/ml) was dissolved in isolation buffer by slowly heating on a hot plate stirrer until the solution was clear, then the digitonin was cooled on ice. Digitonin was added to the microsomal preparation (0.2 mg/mg protein) with stirring at 4°C. Three minutes after the addition of the digitonin, 3 vol of isolation buffer was added and the solution was centrifuged for 20 min at 16,000g. The supernatant was removed and the mitochondrial pellet was resuspended in buffer A with the continuous flow through homogenizer, and then centrifuged again at 16,000g for 20 min. This step was repeated, and the final pellet was resuspended in buffer A with a hand held Teflon homogenizer, to a final concentration of approximately 20-50 mg protein/ml. The mitochondria were then frozen in a large Erlenmeyer flask that was precooled by swirling it in liquid nitrogen. Once the slurry was frozen in liquid nitrogen, it was stored at -8O’C for up to 3 months with no loss in activity. The mitochondria were thawed by removing the Erlenmeyer from the -80°C freezer and warming the contents slowly at room temperature. Once the mitochondrial slurry began to thaw, protease inhibitors were added with constant swirling until completely thawed. The solution of inhibitors consisted of 0.8 vol of buffer B, 0.1 vol of buffer C, and 0.001 vol of (Z-Phe-Phe) and (Z-Phe-Ala) (10 mM in DMSO), and 0.5 mM PMSF. After the pellet was thawed, the solution was kept at 4°C throughout the remainder of the purification. The freeze-thaw solution was centrifuged at 20,400g for 30 min with the brake at MAX. This pelleted the mitochondrial inner membrane (which was discarded) and the matrix enzymes were released in the supernatant (freeze-thaw supernatant). With stirring, 50% polyethylene glycol (PEG 6000mw) was added to the freeze-thaw supernatant to a final concentration of 2% (v/v). After stirring at 4°C for 20 min, the mixture was centrifuged for 10 min at 20,400g. The PEG pellet was discarded, and additional PEG was added to the supernatant to a final concentration of 4%. After another 20.min stirring on ice, the centrifugation step was repeated and the 4% pellet was resuspended in buffer D using a motor driven Teflon homogenizer (760 rpm) to obtain a final concentration of approximately 3 units branched chain complex/ml. The solution was then stored at -8O”C, or applied to the hydroxylapatite column. The solution was clarified by centrifugation (19,OOOg, 10 min) before it was applied to the hydroxylapatite column.
BRANCHED I
I
I
I
CHAIN 1 *
COMPLEX
3
KINETICS
chain complex was consistent with a three-site ping-pong mechanism (6). In the absence of products, the rate equation for a three-site ping-pong reaction mechanism with two ping-pong sites (6) can be reduced to:
V Ka Kb l+[Al+[Bl+[Cl
ll=
I
I
II
30
I
I
50 70 KoAI-‘, (mu)-’
I
90
FIG. 1. Double reciprocal plot for CoA versus KIV. Initial velocity pattern when CoA was varied at fixed concentrations of added KIV of 0.50 (O), 0.125 (O), 0.042 (X), and 0.031 mM (B). NAD was held constant at 1 mM, TPP at 0.2 mM, and lipoamide concentration was 0.061 units/ml.
The clarified PEG supernatant (up to 15 mg protein) was layered on a hydroxylapatite column at a flow rate 0.25 ml/min (larger columns were used for larger protein preparations and the flow was increased accordingly so that the bed volume of the column could be washed every 30-45 min) . After all of the protein had entered the column, the protein was washed with degassed 140 mM potassium phosphate buffer containing 1 mM DTT and 0.2 mM TPP, pH 7.3, until the initial protein peak (which contained the pyruvate dehydrogenase complex) was eluted, and no more pyruvate dehydrogenase activity could be assayed (the pyruvate dehydrogenase assay was identical to the branched chain complex assay, except the ketoacid substrate was pyruvate) Following the complete elution of the pyruvate dehydrogenase complex a linear gradient of phosphate buffer was then applied to the column (140-560 mM phosphate, 1 mM DTT, and 0.2 mM TPP, pH 7.3) over a 2- to 4-h period and at a flow rate of 0.25-1.25 ml/min (depending on the size of the column) with a programmable Ultrograd gradient maker (LKB) and constant flow pump (Isco) . An optical density scanner recorded the absorbance of the eluant from the column and allowed us to identify the protein peaks. All protein fractions were collected and assayed for branched chain complex activity. Active fractions were collected (220-280 mM phosphate buffer), and 0.81 units lipoamide/ml active fractions were added to the combined fractions which were then centrifuged in a Beckman Ti 50 rotor at 165,300g for 2 h. The final pellet was resuspended in buffer D, to a concentration of approximately S-10 units/ml. Before freezing, the branched chain complex was clarified by centrifugation at 15,000g for 15 min. The enzyme was then stored in 50200 rl aliquots at -80°C for 2-3 months without any loss of activity. The specific activity of this preparation ranges from 3 to 4 U/mg and was homogeneous on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified branched chain complex samples were also analyzed for protein content (lo), to determine the specific activity of the branched chain complex. BSA was used as the protein standard in the assay. The nomenclature used throughout the kinetic analysis is that described by Cleland (7).
RESULTS
The first series of experiments was designed to determine whether or not the overall reaction of the branched
Kc ’
where K,, Kb, and Kc are the Michaelis constants for the ketoacid, CoA, and NAD+ , respectively (3 ) . Both the lipoyl dehydrogenase and the ketoacid decarboxylating sites are assumed to be ping-pong ( 6). Initial velocity patterns obtained when one substrate concentration (KIV or CoA) was varied in the presence of a series of fixed concentrations of a second substrate ( CoA or NAD+) resulted in double reciprocal plots with a series of parallel lines, consistent with a ping-pong mechanism (Figs. 1 and 2). Since these results were consistent with the ping-pong mechanism, it was not necessary to vary KIV at a series of fixed concentrations of NAD+. The kinetic constants for ketoisovalerate, ketoisocaproate, ketomethylvalerate, CoA, and NAD+ were 18.3 1- 2.1, 16.7 f 1.4, 10.5 + 1.5, 10.5 XL 2.0, and 49.3 f 6.5 PM, respectively. Inhibition of the reaction by product inhibitors of the branched chain complex were also investigated. In a threesite ping-pong mechanism, products binding at the same site the substrate binds were expected to exhibit competitive inhibition patterns. Furthermore, products binding to other substrate binding sites were expected to exhibit uncompetitive patterns, since the individual site reactions were ping-pong (Figs. 1 and 2). NADH and isovaleryl CoA inhibited branched chain complex activity competitively with respect to their sub-
,
I
I
I
I .
.36 -i
1
3
12 NAZI:-‘, (mM) ”
FIG. 2. Double reciprocal plot pattern when NAD was varied were 0.10 (0). 0.033 (O), 0.02 constant at 1 mM, TPP at 0.2 0.061 units/ml.
for NAD+ versus CoA. Initial velocity at fixed concentrations of added CoA (X), and0.011 mM (m). KIV was held mM, and lipoamide concentration was
4
BOYER
AND
ODESSEY
1
I I
I -2
I 1 4 MADl-B
!I 1
1 l2
I 16
(Id) -’ FIG. 3. Product inhibition NAD. The concentrations of (X), and 0.03 mM (m). KIV, mM, respectively. Lipoamide
by NADH at varying concentrations of added NADH were 0 (0)) 0.01 (O), 0.02 TPP, and CoA were held at 1, 0.2, and 1 concentration was 0.061 units/ml.
strate precursors (Figs. 3 and 4). In addition, isovaleryl CoA and NADH both inhibited the branched chain complex uncompetitively with respect to KIV (Figs. 5 and 6). However, NADH versus CoA (Fig. 7) and isovaleryl CoA versus NAD+ (Fig. 8) did not conform to the expected inhibition pattern. Inhibition of the branched chain complex by isovaleryl CoA versus CoA at nonsaturating NAD+ concentrations (0.125 and 0.060 mM) maintained a competitive inhibition pattern (data not shown). Secondary plots (slope replots) of slope versus isovaleryl CoA concentration were also linear. With decreasing NADf concentrations the Kzp for CoA, Kpp for isovaleryl CoA, and the V zp decreased (Table I), as would be expected with a ping-pong mechanism (2). However, the K”,pP/ V “,pp ratio decreases as
T
.36 -32 t -&- .28 E .24 1 -20 T - .16 i .12 .08 -04
2
I
I
1
40
20
lo
I I 50
[KM-I,‘irnM)-I FIG. 6. Product inhibition by isovaleryl CoA versus KIV. The concentrations of added isovaleryl CoA were 0 (0)) 0.05 (0) , and 0.10 mM ( X ) . NAD, TPP, and CoA were held at 2,0.2, and 0.05 mM, respectively. Lipoamide concentration was 0.061 units/ml.
the NAD+ concentration decreases and is inconsistent with a ping-pong mechanism [ ping-pong mechanism predicts no change in the Kzp/ V zp ratio, (2)]. The competitive inhibition pattern for NADH versus NAD’ at nonsaturating CoA concentrations (0.033 and 0.016 mM) was maintained. Unexpectedly, the slope replots were parabolic (Fig. 9). The K$P for NAD+, K?P for NADH and V $‘P decreased as the CoA concentration decreased (Table I). These observations cannot be explained by the ping-pong mechanism formulated for the other ketoacid dehydrogenase complexes (6). DISCUSSION
Although some kinetic properties of the branched chain complex have been elucidated (11-14)) this early work represents a collection of data obtained from partially purified enzyme preparations and several different cofactor concentrations were used. To our knowledge, a com-
I ’
1
I
I
I
I
f= I
I
-20
I lo
I
30
I
--I
90 wl
FIG. 4. Product inhibition of CoA. The concentrations (O), 0.02 (X), and 0.03 mM 0.2, and 1 mM, respectively. ml.
I
-?(mM)-~”
by isovaleryl CoA at varying concentrations of added isovaleryl CoA were 0 (0) , 0.01 (W). KIV, TPP, and NAD were held at 1, Lipoamide concentration was 0.061 units/
I
I
2
la
I
20
I
1
I
30
40
50
[KM-‘, (mu)-’ FIG. 6. Product inhibition by NADH versus KIV. The concentrations of added NADH were 0 (0)) 0.01 (O), and 0.03 mM (X ) . NAD, TPP, and CoA were held at 0.8, 0.2, and 0.05 mM, respectively. Lipoamide concentration was 0.061 units I ml.
BRANCHED
& 5
0.4
2 z
0.3
CHAIN
COMPLEX
5
KINETICS
0.1
FIG. 7. Product inhibition by NADH versus CoA. The concentrations of added NADH were 0 (0)) 0.0125 (0)) and 0.025 mM (X). NAD, TPP, and KIV were held at 1, 0.2, and 1 mM, respectively. Lipoamide concentration was 0.061 units/ml.
FIG. 8. Product inhibition by isovaleryl CoA versus NAD+. The concentrations of added isovaleryl CoA were 0 ( 0 ) , 0.01 (0 ) ,0.02 mM ( X ) , and 0.03 mM (m) . CoA, TPP, and KIV were held at 0.1,0.2, and 1 mM, respectively. Lipoamide concentration was 0.061 units/ml.
plete steady state kinetic analysis of the branched chain complex has not been performed prior to this investigation. Our experimental design was based on the work of Tsai et al. (3)) who performed the first steady state kinetic analysis of the pyruvate dehydrogenase complex using recently developed rate equations for a three-site pingpong mechanism (6). We have also correlated the branched chain complex initial velocity and product inhibition patterns with patterns predicted by the rate equations derived by Cleland for a three-site ping-pong mechanism. Inhibition by NADH versus NAD+ and isovaleryl COA versus CoA was competitive, while the inhibition by NADH or isovaleryl CoA versus KIV was uncompetitive, as predicted by the rate equations. These patterns were
similar to those reported for the pyruvate dehydrogenase (3) and a-ketoglutarate complex (4). Exceptions to the patterns predicted by the rate equations, present in all three ketoacid dehydrogenase complexes [ (3, 4) and present study], were the inhibition patterns observed for isovaleryl CoA versus NAD+ and NADH versus CoA, which were predicted to be uncompetitive. NADH versus CoA resembles a mixed type of inhibition with the lines intersecting in the third quadrant. The reciprocal plot of isovaleryl CoA versus NADf yields a set of lines which fail to converge in a single quadrant. These results could not be compared to the data of Tsai et al. (3) or Hamada et al. (4)) since they only used one inhibitor concentration. Cleland (6) has attempted to explain the unexpected inhibition patterns obtained by Tsai et al. ( 3 ) . Noncom-
TABLE Kinetic Competitive inhibitor Isovaleryl-CoA
NADH
Fixed [substrate] NAD (1 mM) (0.125 mM) (0.060 mM) CoA (1 mM) (0.1 mM) (0.033 mM) (0.017 mM)
Constants
for
Nonsaturating
I Product
Inhibition
Varied substrate KfP CoA 7.4 f 1.0 3.7 k 0.7 1.6 + 0.4 NAD 46.9 rfr 49.3 + 42.7 f 34.0 f
5.3 6.5 5.0 6.0
Slope replots
10.5 f 2.0 2.5 f 0.4 1.1 f 0.3
10 + 0.3 7.5 f 0.2 4.2 k 0.1
0.75 0.49 0.38
1.42 0.68 0.69
Linear -
10.2 12.7 9.7 6.7
14.4 16.3 11.9 11.1
3.26 3.02 3.59 3.12
0.22 0.26 0.23 0.20
Parabolic
* + f +
1.5 2.5 1.0 1.0
f f f +
0.3 0.5 0.3 0.4
Note. Product inhibition experiments were carried out as described in the methods and illustrated in Figs. 3 and 4. The Kzp, Kz!‘, and Vz*, are apparent Michaelis constants (K, app for the varied substrate, KT” for the inhibitor, and Vzp for the velocity expressed in nmol NADH formed per minute). All Michaelis constants were calculated using the nonlinear regression as described in the methods. The KIV and TPP concentration was held constant at 1 mM and the lipoamide concentration The isovaleryl CoA and NADH concentration ranged from 0.005 to 0.3 mM. All constants are listed as means f S.D. and are
-
abbreviations used, apparent maximal program Pennzyme was 0.061 unit/ml. in (PM).
6
BOYER
AND
7 5 0
4
8
12
16
20
24
28
FIG. 9. Secondary plot of the slope versus inhibitor concentration for inhibition by NADH at fixed CoA concentrations. The CoA concentrations were 0.1 (+ ) , 0.033 (O), and 0.016 mM (Cl). KIV was held constant at 1 mM, TPP at 0.2 mM, and lipoamide concentration was 0.061 units/ml.
petitive inhibition patterns were observed for acetyl CoA versus NAD+ and for NADH versus CoA in the pyruvate dehydrogenase complex (3). He concludes that a product will be noncompetitive against the substrate that binds at the next site in the overall reaction sequence if that site is random sequential. In addition, if a product combines as a dead-end inhibitor at another site, or hinders the reaction at this other site, it will be noncompetitive versus the substrate that normally binds at that site (3). In contrast to the observations of Tsai et al. (3) and Hamada et al. (4)) we observed a mixed type of inhibition for NADH versus CoA (Fig. 7) and a set of lines which fail to converge in a single quadrant for isovaleryl CoA versus NAD+ (Fig. 8). To further explain these anomalous patterns Tsai et al. (3) investigated the effect of pyruvate and CoA on theflavoprotein-catalyzedreaction (lipoamide + NADH + H+ + dihydrolipoamide + NAD+) . CoA, but not pyruvate, inhibited the complexed flavoprotein (El-E2E3) noncompetitively with respect to NADH and lipoamide ( 3 ) . In addition, noncompetitive inhibition patterns were observed with CoA versus NAD+ and CoA versus dihydrolipoamide ( reverse reaction), while CoA and pyruvate failed to inhibit the uncomplexed protein. Two explanations were proposed on the basis of these findings ( 3 ) : (i) The first possibility is that the transacetylase (E2-core) and flavoprotein (E3) catalytic centers are so close that binding of CoA (or acyl CoA) to E2 sterically hinders binding of NADH (or NAD+) to E3, and visa versa. (ii) The second possibility is that the binding of CoA (or acyl CoA) to E2 causes a conformational change in E2 that hinders binding of NADH (or NAD+) to E3.
ODESSEY
It appears that all three intramitochondrial multienzyme complexes have similar kinetic E2-E3 interactions. This is not surprising since the E3 component of all three complexes is thought to be identical ( 1). We attempted to describe the interaction of two potential physiological feedback inhibitors (NADH and isovaleryl CoA) by studying their inhibition patterns at nonsaturating concentrations of “fixed” substrate (the fixed substrate binds at a different site than the substrate being varied). Rate equations for a ping-pong mechanism state that the ratio of Kzp/ V “,pp is independent of the fixed substrate concentration and, therefore, should not change as the concentration of fixed substrate is reduced (2). A fixed KzP/VzP ratio is predicted for a ping-pong reaction because the apparent Michaelis constants increase to their limiting values as the fixed substrate concentration increases, and decrease as the fixed substrate concentration decreases. For a sequential reaction however, the Michaelis constants may change in any direction as the fixed substrate concentration increases, since the change in Michaelis constant depends on whether the Ki, is less than, greater than, or equal to the K, (2 ) . The apparent V,,, is the same for both types of reactions. Therefore, as the fixed substrate concentration increases, the K”,pp for a sequential reaction must decrease or remain constant (2). As seen in Table I, the K$P for CoA increases as NAD+ (fixed substrate) increases and the Kzp for NAD+ increases as the CoA concentration increases. This suggests that both reactions are indeed pingpong. The fixed substrates were varied from nonsaturating to saturating concentrations (Table I). For the inhibition of the branched chain complex by NADH, the K$‘P/VampP ratio does not change (Table I). Conversely, the Kzp/ V “,ppratio for inhibition by isovaleryl CoA at fixed NAD+ concentrations does tend to decrease. This observation does not confirm a sequential or ping-pong step in catalysis (2). We also observed a increase in the K!PP/ Kzp ratio (isovaleryl CoA/CoA) with increases in the level of fixed second substrate (NAD+) . In contrast to these results, Smith et al. ( 5) performed similar product inhibition studies with a-ketoglutarate dehydrogenase and found that as NAD” levels were increased, the KqPP/ Kzp for succinyl CoA versus CoA remained constant. Although the slope replots for the inhibition of the branched chain complex by isovaleryl versus CoA were linear, replots obtained for NADH versus NAD+ were parabolic. If an inhibitor binds at two different mutually exclusive sites, a linear mixed type of inhibition can occur and the system resembles a mixture of pure competitive and pure noncompetitive inhibition ( 15). This type of inhibition results in a failure of the reciprocal plots to converge at a common point and slope replots are parabolic. NADH has consistently been observed to be a pure competitive inhibitor against NAD; however, binding of NADH to an additional noncompetitive
BRANCHED
CHAIN
site has not been reported to our knowledge. It is therefore possible that a linear mixed type of inhibition does not account for the observed inhibition pattern. Further experiments are required to define this complex inhibitory pattern. In conclusion, the data we have obtained on the steady state kinetics of the branched chain complex correspond well to a three-site ping-pong mechanism (6). The exceptions include the inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA. Interestingly, we have observed a linear mixed type of inhibition pattern for NADH versus NAD + at nonsaturating CoA concentrations. To our knowledge, this has not been reported for the pyruvate dehydrogenase or a-ketoglutarate dehydrogenase complexes. Although subtle differences in the catalytic mechanism of the branched chain complex, pyruvate dehydrogenase complex, and ol-ketoglutarate dehydrogenase complex exist, these three multienzyme complexes appear to fit a three-site ping-pong mechanism. It is clear that further work is necessary to more precisely define their catalytic mechanisms. This kinetic study of the branched chain complex affords a better understanding toward this goal.
COMPLEX
KINETICS
7
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