Plant Science, 71 (1990) 159--166
Elsevier Scientific Publishers Ireland Ltd.
Kinetic mechanism of potato tuber succinate semialdehyde dehydrogenase Vaduvatha Satya Narayan and P. Madhusudanan Nair* Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Bombay 400 085 (India) (Received February 27th, 1990; revision received June 4th, 1990; accepted June 7th, 1990)
The steady state kinetic mechanism of purified succinate semialdehyde dehydrogenase enzyme from potato (Solarium tuberosum L. cv Kufri Chandramukhi) tubers was investigated. The potato enzyme exhibited Michaelis-Menten kinetics with respect to both the substrates, succinate semialdehyde and NAD'. Initial velocity studies suggested a sequential mechanism for the enzyme. The enzyme had a relatively low K value for succinate semialdehyde (4.65/aM) and was inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD ÷(K~ = 215/aM). NADH, one of the products of the reaction, inhibited competitively with respect to NAD ÷ with a K~value of 53.8/aM and non-competitively with respect to succinate semialdehyde, p-Hydroxybenzaldehyde, as a dead-end inhibitor, gave competitive inhibition with respect to succinate semialdehyde and uncompetitive inhibition with respect to NAD ÷. The results of these studies, together with analysis of the dead-end inhibition by AMP, were consistent with an ordered bi, bi sequential mechanism for the potato enzyme in which NAD* was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it. The mechanism obtained here was compared with the mechanisms proposed for this enzyme from other sources.
Key words: potato; enzyme; semialdehyde dehydrogenase; kinetics; mechanism
SSADH catalyses the pyridine nucleotide dependent oxidation of SSA to succinate [I]. It is the last enzyme of the Abu shunt pathway [2--4] which provides an alternative route for glutamate to enter the tricarboxylic acid cycle. The enzyme has been purified from several microorganisms [5--9] and mammalian brain [10--15] and characterized in detail. Steady state-initial velocity studies of brain SSADH from various sources have resulted in the proposal of conflicting kinetic mechanisms [10--12]. As in bacteria [1,5--9] and animals [2--4], Abu is metabolised in higher plants by the operation of *To whom correspondence and reprint requests should be addressed. Abbreviations: Abu, 4-aminobutyrate; p-OHB, p-hydroxybenzaldehyde; SSA, succinate semialdehyde; SSADH, succinate semialdehyde dehydrogenase (suecinate semialdehyde : NAD* oxidoreductase (EC 184.108.40.206).
the Abu shunt [16--18]. Although the first two enzymes of the Abu shunt, glutamate decarboxylase and Abu transaminase have been well characterized from higher plants [16,18,19], there is very limited data concerning SSADH. The presence of SSADH has been shown in crude extracts prepared from radish cotyledons , wheat embryos [201 and barley seeds  and the enzyme has been purified from the last two sources. However all the previous studies on plant SSADH employed Tris--HC1 buffer and high concentrations (> 0.5 mM) of substrate, SSA, for the enzyme assay, conditions that are reported to inhibit severely the brain enzyme from various species [10--15]. In addition, no information is available on the kinetics of SSADH from a higher plant. As part of our detailed studies on the metabolism and enzymology of Abu in higher plants [18,19,22], we have recently reported the purification and physical characterization of SSADH from potato tubers  where we have shown that
0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
160 potato enzyme is inhibited by Tris and high concentrations of SSA. We now report the kinetics of homogeneous potato tuber SSADH under noninhibitory assay conditions. Materials and Methods
Plant material Potato tubers (Solanum tuberosum, Kufri Chandramukhi cultivar) were obtained from the local market within 1 month after harvest and were stored at room temperature during the studies. Chemicals SSA, NAD ÷, NADH, 3-acetylpyridine adenine dinucleotide, A M P and p-hydroxybenzaldehyde were obtained from Sigma. All other chemicals were of highest analytical grade, p-Hydroxybenzaldehyde was recrystallised from water before use. SSA obtained from Sigma was purified before use to remove polymeric and oxidised material by the method of Taberner et al. . Substrate inhibition studies were carried out with SSA synthesized in our laboratory by the Bruce et al.  procedure. The actual concentration of SSA monomer in solution was determined enzymaticaUy by the GABASE method . Purification Purification of SSADH from potato tubers was carried out exactly as described previously . The final preparation from the 5'AMP-sepharose4B affinity chromatography step was made 25°7o (v/v) with respect to glycerol and stored at - 2 0 ° C . Glycerol was removed from the samples on the day of use by dialysis against 100 mM sodium pyrophosphate buffer, p H 7.0 containing 14 mM 2-mercaptoethanol. The enzyme was homogeneous as judged by polyacrylamide gel electrophoresis, both in the presence and absence of sodium dodecyl sulphate and by chromatofocussing . The specific activity of the enzyme used in the present studies was in the range of 6.2 --6.5 tamol (mg protein) -~ min -~ at 25 °C. Enzyme assays SSADH activity was determined spectrophoto-
metrically at 340 nm at 25 °C with a Shimadzu UV 240 double beam recording spectrophotometer. In 3.0 ml, the reaction mixture contained 100 mM sodium pyrophosphate buffer, p H 9.0, 14 mM 2mercaptoethanol, 0.5 mM NAD ÷ and enough enzyme to give an absorbance change of 0.05-0.1/min. After equilibration for 5 min, the reaction was initiated by the addition of I00 taM SSA (final concentration) to the experimental cuvette. Any variations in this procedure are described in the figure legends. One unit of enzyme is defined as the amount required to produce 1 tamol NADH/min at 25°C. The enzyme reaction was found to be proportional to the protein concentration under all the assay conditions employed.
Protein determ&ation Protein determinations were done by the Bradford method  using bovine serum albumin as standard. Inhibition studies All compounds used for inhibition studies were dissolved in 100 mM sodium pyrophosphate buffer (pH 9.0). The pH of the assay mixture, after addition of all components, was adjusted to pH 9.0. Each inhibitor was incubated with the enzyme in buffer for 10 rain at 25 °C before initiating the reaction by the addition of substrate. Data analysis The points shown in all figures are the means of three (in case of low absorbance, four) independent determinations. Data points were fitted to the best line in Lineweaver-Burk space by utilizing a least squares linear regression analysis. Kinetic parameters were determined from secondary slope a n d / o r intercept replots. The nomenclature used in the analyses of kinetic data is that of Cleland
The reaction of potato SSADH, like the enzyme from other sources [12,15], is irreversible since succinate was unable to oxidise N A D H even after long periods of incubation. This irreversibility and failure of one of the products, succinate,
to inhibit the reaction limited the steady state kinetic investigation o f the enzyme since the full range o f product inhibition and isotope exchange experiments that usually allow distinctions among many mechanisms to be made are excluded. Rivett and Tipton  have employed 3-acetylpyridine adenine dinucleotide as an alternative coenzyme to delineate the kinetic mechanism of rat brain SSADH. Lack of activity of potato enzyme with this alternative enzyme further hampered our work. However, the kinetic mechanism of potato SSADH was examined using initial velocity studies with both the substrates and by experiments involving inhibition by substrate, product and dead-end inhibitors. The results of these studies eliminated certain mechanisms and allowed us to arrive at the possible pathway(s) followed by the potato enzyme.
Initial velocity studies Potato SSADH exhibited normal, MichaelisMenten type o f kinetics with respect to both the substrates. Double reciprocal plots of 1/v against 1/(SSA) at a series of concentrations of NAD ÷ gave a series of straight lines intersecting above the -1/(SSA) axis (Fig. 1). Similar results were
obtained when a double reciprocal plot o f I/v against 1/(NAD ÷) at a series o f concentrations o f SSA was analysed (Fig. 2). These observations suggested a sequential mechanism of substrate addition for the enzyme which can be described by the general equation
+ KJ(A) + K / ( B ) + (A)(B) The values o f the constants in the equation were calculated from the slope and intercept replots as V (maximal velocity) = 6.26 units/mg; K (Michaelis constant for NAD ÷) = 31.3 /aM, Kia (dissociation constant for enzyme-NAD + complex) = 167.8/~M and K b (Michaelis constant for SSA) = 4.65/aM. When the substrates were mixed in a fixed concentration ratio, the double reciprocal plot of V against the concentration o f this mixture was nonlinear (Fig. 3) which confirmed the suggestion of the sequential nature of substrate addition. In the case of a ping pong mechanism, this plot would be a linear one .
Substrate inhibition Potato SSADH was inhibited by the substrate, SSA, at concentrations above 120/aM. The inhibi-
~ 8o GO
-10 0 1C
~f.~uCCINIC SEMIALDEHYD] mM"1
Fig. 1. Double reciprocal plot of (SSA) against the initial velocity of potato tuber SSADH. Assays were carried out in 0.1 M sodium pyrophosphate buffer pH 9.0 containing 14 mM 2mercaptoethanol at 25 °C. (NAD ÷) are: O, 8/aM; A, 16/aM; El, 40 gM; O, 80 gM.
30 ~ /.0 5 0 - I 60 I / I N , O'J m M
Fig. 2. Double reciprocal plot of (NAD +) against the initial velocity of potato tuber SSADH. (SSA) are: O, 1/aM; A, 1.75 /aM; [3, 2.5/aM; o , 5.0/aM.
I/CSUBSTRATE MIXTURE] mM-I Fig. 3. Double reciprocal plot for potato tuber S S A D H with the substrates varied in a fixed concentration ratio. The substrates were mixed in the ratio of the K concentration of SSA: 2.5 times the K mconcentration of NAD +.
tion was linear, i.e. l / v became linear with (SSA) and was competitive with respect to N A D ÷ with a K i value o f 215/~M (Fig. 4).
Product inhibition 10 m M succinate did not affect the potato enzyme significantly. Inhibition by the other product, N A D H , was competitive with respect to N A D ÷ at non-saturating concentrations o f SSA with a K i value o f 53.8/~M (Fig. 5A). The replot o f
1 I 2 3 [$UCClmito smc~kll~le~
I 4 mM -1
High substrate inhibition of potato tuber SSADH. (NAD°) are: O, 20/~M; A, 30/~M; [22,40/~M; O, 80/~M. Fig. 4.
4 8 12 I/[NAD+] rnM -I
-20 0 2b 20 s'0 do I/[succinate semialdehyde] mM-!
Product inhibition of potato tuber S S A D H
NADH. (A) (NAD +) was varied with a fixed concentration of SSA at 5 ~M. (NADH) are: O, 0; A, 25 ~M; [3, 50/JM; e , 100 /~M. (B) NAD ÷concentration was fixed at 32/~M. (NADH) are: O, 0; A, 25 ~M; 1-1,75/~M; o , 150/~M.
slopes against ( N A D H ) was linear (data not shown) suggesting that the inhibition was o f the linear competitive type . N A D H inhibition with respect to N A D ÷ at saturating concentrations o f SSA could not be determined accurately owing to interference by high substrate inhibition. Inhibition by N A D H was found to be non-competitive with respect to SSA at a non-saturating concentration o f N A D ÷ (Fig. 5B). Slope and intercept replots were linear. N o inhibition by N A D H was observed at a saturating concentration o f N A D ÷.
Studies with dead-end inhibitors The effects o f 5'AMP and p-hydroxybenzaldehyde as dead-end inhibitors were studied . 5'AMP was found to be a competitive inhibitor with respect to N A D +, with a K value o f 269 /~M (Fig. 6A) and non-competitive with respect to SSA (Fig. 6B). Inhibition was linear in both the cases as determined by slope and intercept plots. The inhibition by p-hydroxybenzaldehyde was competitive with respect to SSA with a K i value o f 2.9/~M (data not shown) and uncompetitive with respect to N A D ÷ as shown in Fig. 7. In an ordered mechanism, a competitive inhibitor for the first substrate A will give non-competitive inhibition with respect to substrate B. On the other hand, a
inhibitor for the other substrate . Uncompetirive inhibition by p-hydroxybenzaldehyde with respect to N A D + thus strongly suggests that the enzyme follows an ordered sequential mechanism of substrate addition, p-Hydroxybenzaldehyde was found to have no significant effect on the K~ value for high substrate inhibition by SSA.
B tll 80
/ I I,
I / [ . , o ~ ] . , . -~
Fill. 6. Inhibition of potato tuber SSADH by AMP. (A) SSA concentration was fixed at 10/aM. (AMP) are: O, 0; A, 0.3 mM; I"1, 1 mM; O, 1.4 mM. (B) NAD÷concentration fixed at 32/aM. (AMP) are: O, 0; A, 0.15 raM; El, 0.5 raM; o, 0.7 mM.
dead-end competitive inhibitor for substrate B would be expected not to react with free enzyme to which (A) binds but rather with the E A binary complex and this will yield uncompetitive inhibition with respect to substrate (A). In the case o f a r a n d o m mechanism, a competitive inhibitor for either substrate will act as a non-competitive
:::P I -s
Fig. 7. Inhibition of potato tuber SSADH by p-hydroxybenzaldehyde. SSA concentration was fixed at 10 /aM. (pHydroxybenzaldehyde)are: O, 0; A, 2.5/aM; El, 5/aM; e , 10 /aM.
Intersecting patterns of straight lines for double reciprocal plots (Figs. 1 and 2) are consistent with a sequential mechanism for the addition of substrates. A substituted or ping-pong mechanism is ruled out by this pattern and by the non-linearity o f the dependence obtained when the two substrates were varied in a fixed ratio (Fig. 3) . When two substrates A and B are maintained at a constant ratio, A = a(B) where a is a constant. Substitution of this relationship into the rate equation according to Dalziel notation gives 1/v = ~o + t J A / A + aiJB/A for a ping pong mechanism and 1/v = ~o + ~ A / ( A ) + a ~ B / ( A ) + a ~ A B / (A 2) for a sequential mechanism . When 1/v is plotted against 1/(A), the plot will be linear with an intercept = I~0 for a ping pong mechanism. In a sequential mechanism, the substrate squared term makes the plot parabolic up with a minimum in the second quadrant to the left o f the 1/v axis. The linearity of the plots shown in Figs. 1 and 2 also make it unlikely that a r a n d o m order steady state mechanism is obeyed. The point of convergence of the double reciprocal plot of v against (SSA) rules out an equilibrium ordered mechanism in which case the lines on the 1/(SSA) versus 1/v plot would intersect on the 1/v axis indicating a value of 0 for ~A . Thus initial velocity studies suggested that the potato enzyme m a y follow either a steady state ordered mechanism or a rapid equilibrium random mechanism. In a random mechanism, one does not expect substrate inhibition except where one substrate shows some affinity for the site of another, and this should result in competitive substrate inhibition . Thus uncompetitive inhibition of potato SSADH by SSA with respect to N A D + (Fig. 4) is more in agreement with a compulsory order mechanism involving N A D H being the last product to
dissociate from the enzyme and inhibition resulting from the formation o f an abortive ternary complex between enzyme, N A D H and SSA. Linear inhibition by substrate indicates that the inhibition was due to binding of only a single molecule of SSA to an enzyme form (such as E - N A D H ) with which it was not supposed to bind. If two molecules of SSA were to combine with the enzyme form with which it normally binds, but in a dead-end fashion, then substrate inhibition would have been parabolic [321. The linear competitive nature of the N A D H inhibition with respect to N A D ÷ (Fig. 5A) indicates that both NAD* and N A D H might be binding to the same form of enzyme, i.e. free enzyme. On the other hand, non-competitive inhibition by N A D H with respect to SSA (Fig. 5B) indicates that SSA binds to a f o r m different f r o m the one to which N A D H was binding. However, at saturating levels of N A D ÷, all the free enzyme would be present as the E - N A D ÷ complex and hence N A D H would not be able to bind to the free enzyme. In this situation, reversible connections between the various forms of enzyme are broken and therefore no inhibition is expected. The absence of inhibition by N A D H at a saturating concentration of N A D ÷ is in support of this argument. Uncompetitive inhibition by p-hydroxybenzaldehyde with respect to NAD* (Fig. 7) can be explained only in terms of a compulsory ordered mechanism and not by a r a n d o m mechanism. In the case of a r a n d o m mechanism, inhibition by phydroxybenzaldehyde with respect to N A D ÷ would be non-competitive . The lack of any effect of p-hydroxybenzaldehyde on the high substrate inhibition by SSA shows that no significant binding of this c o m p o u n d to the E - N A D H complex occurs under these conditions. All these results are thus consistent with a compulsory ordered bi, bi-mechanism in which N A D ÷ binds before SSA and N A D H is the last product to dissociate from the enzyme. It is, however, not possible from the presently available data to distinguish between a mechanism involving a ternary complex and a Theorell-Chance mechanism. These two possible mechanisms showing the interactions with the dead-end inhibitors and with SSA to give high substrate inhibition are shown in
Scheme 1. In both cases, it is assumed that the release of succinate is essentially irreversible to account for the lack of detectable inhibition by this product. (a) Mechanism involving ternary complex formation: E ~
E-NAD ~ ~ E-NAD+-SSA ~ E-NAD-Succinate
E-NADH ~ E
(b) Theorell-Chance mechanism: E ~ E-NAD ÷ It E-AMP
Possible kinetic mechanisms of potato SSADH.
In their studies Blaner and Churchich  could find no evidence for the formation of an enzyme-NADH-succinate complex in the case of the brain enzyme and thus it is possible that the ternary complexes are not kinetically significant which would be indicative of the Theorell-Chance mechanism rather than that involving ternary complexes. However, it is likely that the brain and potato enzymes have different mechanisms and the later enzyme m a y form kinetically significant ternary complexes. The mechanism proposed here for the potato enzyme differs f r o m the ordered ter-bi mechanism proposed by Cash et al.  for the rat brain enzyme. These workers reported uncompetitive cosubstrate effects rather than non-competitive as reported here and by others [11,12] but they found an unusually high K value for SSA and did not observe high substrate inhibition below 1 mM. Since their experiments were carried out in Tris buffer, it may be difficult to interpret them. In contrast to our results and those obtained by others [10,I 1], Blaner and Churchich  found that inhibition of pig brain enzyme by p-hydroxybenzaldehyde was non-competitive with respect to N A D ÷ (although the data were not shown) and concluded that the mechanism was r a n d o m in order to account for this. However, our studies
are in excellent agreement with the results obtained in the case o f rat brain enzyme by Rivett and Tipton . High substrate specificity o f S S A D H together with an extremely low K m value for substrate, in the region o f 2 - - 5 / ~ M , ensures the rapid and efficient conversion o f SSA to succinate and thus prevents the accumulation o f toxic aldehyde in the cells. This is also supported by the failure o f several workers [19,34] to detect free SSA in tissues despite the use o f highly sensitive enzymatic methods o f assay. Thus high substrate inhibition o f S S A D H observed in vitro may be largely a nonphysiological p h e n o m e n o n never encountered in cells. Presence o f high S S A D H activity in tissues where the Abu shunt pathway is absent  suggests that this enzyme might have some other function(s) in addition to linking the Abu shunt to the tricarboxylic acid cycle. These functions could possibly include the oxidation o f SSA generated from some source other than Abu or reacting with some metabolite not tested so far. References 1
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