Saccharopine Dehydrogenase SUBSTRATE INHIBITION STUDIES

In the direction of reductive condensation of cY-ketoglutarate and lysine, saccharopine dehydrogenase (W-(glutar-2-yl)-L-1ysine:NAD oxidoreductase (lysine-forming)) is inhibited by high concentrations of a-ketoglutarate and lysine, but not by NADH. NAD+ and saccharopine show no substrate inhibition in the reverse direction. Substrate inhibition by cu-ketoglutarate and lysine is linear uncompetitive uersus NADH. However, when the inhibition is examined with cy-ketoglutarate or lysine as the variable the double reciprocal plots show a family of curved lines concave up. The curvature is more pronounced with increasing concentrations of the inhibitory substrate, suggesting an interaction of variable substrate with the enzyme form carrying the inhibitory inhibition interaction the E.NAD+ 301-307), of

Saccharopine Dehydrogenase SUBSTRATE INHIBITION STUDIES (Received for publication, June 26, 1975) MOTOJI FUJIOKA From the College of Bio-Medical Technology, Osaka University, Toyonaka, Osaka 560, Japan In the direction of reductive condensation of cY-ketoglutarate and lysine, saccharopine dehydrogenase (W-(glutar-2-yl)-L-1ysine:NAD oxidoreductase (lysine-forming)) is inhibited by high concentrations of a-ketoglutarate and lysine, but not by NADH. NAD+ and saccharopine show no substrate inhibition in the reverse direction.
Substrate inhibition by cu-ketoglutarate and lysine is linear uncompetitive uersus NADH.
However, when the inhibition is examined with cy-ketoglutarate or lysine as the variable substrate, the double reciprocal plots show a family of curved lines concave up. The curvature is more pronounced with increasing concentrations of the inhibitory substrate, suggesting an interaction of variable substrate with the enzyme form carrying the inhibitory substrate. These inhibition patterns, the lack of interaction of structural analogs of lysine such as ornithine and norleucine with the E.NAD+ complex (Fujioka, M., and Nakatani, Y. (1972) Eur. J. Biochem. 25,[301][302][303][304][305][306][307], the identity of values of inhibition constants of a-ketoglutarate and lysine obtained with either one as the substrate inhibitor, and the substrate inhibition data in the presence of a reaction product, NAD+, are consistent with the mechanism that substrate inhibition results from the formation of a dead-end E.NAD+ .c~-ketoglutarate complex followed by the addition of lysine to this abortive complex.
A number of pyridine nucleotide-linked dehydrogenases have been shown to be inhibited by high concentrations of the substrates. The substrate inhibition has generally been considered as arising from the formation of a complex of a substrate and an enzyme form which is not supposed to react with. In pyridine nucleotide dehydrogenases, many examples are known in which substrate inhibition is caused by the formation of an abortive enzyme. oxidized coenzyme oxidized substrate or enzyme. reduced coenzyme . reduced substrate complex (l-5). Recently substrate inhibition resulting from the combination of a substrate with central complexes has also been reported (6,7).

It was previously
shown that saccharopine dehydrogenase (W-(glutar-2-yl)-L-1ysine:NAD oxidoreductase (lysine-forming)) which catalyzed a reversible reaction and/or central complexes, they should give uncompetitive patterns with respect to both NADH and noninhibitory substrate. The curved double reciprocal plots in Figs. 2 and 4 show that the mechanism of inhibition is more complex. However, from the dependence of inhibitory effect of a variable substrate on the concentration of a substrate inhibitor, and the linearity of the plots of l/u versus concentrations of a substrate inhibitor at each concentration of a variable substrate, the observed inhibition patterns may most simply be explained as arising from the combination of a variable substrate with the abortive complex(es) carrying a substrate inhibitor.
Substrate Inhibition in Presence of NAD+-Whereas the experiments of Figs. 1 to 4 are consistent with the assumption above, they do not give indication as to whether the inhibition is caused by the combination of a substrate inhibitor with the E.NAD+ or central complexes, or both. These alternatives may partly be distinguished by running the inhibition experiment in the presence of a constant level of an added product, NAD+. In its presence, the binding of a substrate inhibitor to the E.NAD+ complex will cause a slope effect with NADH as the variable substrate, while the binding solely to the central complexes will not. Fig. 5 shows the a-ketoglutarate inhibition in the presence of NAD+. The presence of NAD+ did give a

Substrate
Inhibition by a-Ketoglutarate and Lysine-A previous investigation has shown that saccharopine dehydrogenase is inhibited by high concentrations of a substrate, a-ketoglutarate (8). Substrate inhibition was also noted by lysine at very high concentrations, but no inhibition was observed with NADH up to 0.2 mM (about 8 times the Michaelis constant) at cr-ketoglutarate and lysine concentrations of 0.5 mM and 2.0 mM, respectively.
High concentrations of NAD+ (up to 5.0 mM) and saccharopine (up to 40 mM) showed no inhibition in the forward direction (Reaction 1) at pH 6.8. The possibility that inhibition by either cY-ketoglutarate or lysine was due to inhibitory contaminants was ruled out because precisely the same degree of inhibition was obtained by use of the compounds which had been recrystallized several times. Fig. 1 shows the plots of reciprocal of initial velocities against reciprocal of NADH concentrations at a constant concentration of lysine (1.8 times the Michaelis constant) and several fixed, high levels of cY-ketoglutarate.
As the figure shows, at high a-ketoglutarate concentrations the inhibition was uncompetitive, and the replot of vertical intercepts versus Lu-ketoglutarate was a linear function. When cu-ketoglutarate inhibition was examined with lysine as the variable substrate at a constant level of NADH, the double reciprocal plots gave a family of curved lines concave upward, although at low lysine concentrations the lines were nearly parallel (Fig. 2). The curvature was more pronounced with higher cu-ketoglutarate concentration.
However, when plots were made of l/u uersus a-ketoglutarate concentrations at each concentration of lysine, they were linear at high concentrations of the inhibitor. Values of the apparent inhibition constants for slope (K,,) and intercept (K,,) were determined by fitting the initial velocity data obtained with a-ketoglutarate concentrations of more than 7.5 mM to Equation 2, and were found to be 15.5 & 1.5 mM and 17.6 f 2.0 mM, respectively.

Previous investigations
have shown that the kinetic mechanism followed by saccharopine dehydrogenase is an ordered Ter Bi mechanism (the reverse direction, Reaction 1) and the sequence of addition of substrates is NADH, cu-ketoglutarate, and lysine (8,9,12). In this mechanism substrate inhibition may arise in the following cases. plots is consistent with this idea and excludes the possibility of any partial inhibition.
The discussion above assumed that both cY-ketoglutarate and lysine at high concentrations could form abortive complexes, and the resulting complexes in turn could absorb the other substrate. But the same inhibition patterns will be seen when only one substrate can bind to the E. NAD+ and/or central complexes, and the other adds subsequently to the complex(es). The binding of lysine to the E .NAD+ or central complexes is unlikely from the lack of interaction of lysine analogs with these complexes.
It has been shown that the analogs of lysine such as ornithine, norleucine, and leucine are potent competitive inhibitors of lysine in the reverse direction and produced no inhibition in the forward direction even at concentrations more than 50 times their dissociation constants from the E .NADH . cu-ketoglutarate .analog complex (8). If cr-ketoglutarate binds to the E. NAD+ complex, the product inhibition by this compound in the forward direction should theoretically give a noncompetitive pattern when saccharopine is the variable substrate. The combination with the central complexes would give rise to noncompetitive inhibitions for variable NAD+ and saccharopine.
Under experimental conditions, however, no appreciable slope effect was observed with respect to both NAD+ and saccharopine (12). This could be due to a large dissociation constant of a-ketoglutarate from these complexes (see below).
If an alternate reaction path in which the order of addition of a-ketoglutarate and lysine is reversed (E 4 E.NADH --t E 'NADH .lysine + central complex) operates at high concentrations of lysine and if lysine forms dead-end complex(es) with either E .NAD+ or central complexes, or both, substrate inhibition for which the double reciprocal plots are linear with respect to NADH and curved to a-ketoglutarte would be obtained. In this mechanism, however, the plots of l/u uersus lysine concentrations would not be linear, contrary to the experimental finding (Fig. 4). It is also difficult to visualize nonlinear reciprocal plots when a-ketoglutarate is the substrate inhibitor and lysine the variable substrate, since the concentrations of the latter are kept at low level. Furthermore, the combination of lysine with the E.NAD+ or central complexes may be excluded from reasons mentioned above.
Thus the observed substrate inhibitions may best be interpreted as the result of combination of a-ketoglutarate with the E.NAD+ and/or central complexes and subsequent binding of lysine to the abortive complex(es) inhibiting the dissociation of NAD+ or catalysis. The rate equation for this mechanism may be written as  From these equations, by substituting values for C, Q, K,, KIa, Kb, K,,, K,, and K,, (1.38 mM), the approximate value off was calculated as 0.63. The f value of less than unity indicates that a-ketoglutarate binds only to the E .NAD+ complex, not to the central complexes; otherwise the value should be 1.
Although the inhibition patterns by cu-ketoglutarate and lysine are rather complicated, in the absence of further data, it may thus be assumed that the inhibition results from dead-end 8989 combination of the former with the E .NAD+ complex and the secondary binding of the latter to this abortive ternary complex, and the prevention of dissociation of NAD+. This interpretation is consistent with a previous finding that Lu-ketoglutarate and lysine in the concentration range covered in the current investigation failed to show substrate inhibition when the coenzyme was NADPH instead of NADH. NADPH did support the reaction, although with much less efficiency than NADH, but no appreciable binding of NADP+ to saccharopine dehydrogenase could be demonstrated (9). Therefore, in the reaction with NADPH, the steady state concentration of E.NADP+ complex would be too low to allow the formation of an abortive complex with Lu-ketoglutarate.
It is of interest to note that, while the dissociation constant of a-ketoglutarate from the E .NAD+ .c~-ketoglutarate complex (KJ calculated from the data of Table I is about 60 times as large as that from the E .NADH .a-ketoglutarate complex (KJ, lysine can bind readily to the E.NAD+ .a-ketoglutarate complex with a dissociation constant comparable to its Michaelis constant.
Apparently the conformation of enzyme protein induced by a-ketoglutarate binding is of primary importance in lysine binding.