Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae

Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed by L-alpha-aminoadipate-delta-semialdehyde ( L-AASA) which adds in rapid equilibrium prior to l-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs alpha-AASA and L-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, alpha-AASA, and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine, L-glutamate is noncompetitive vs both NADP and saccharopine, while L-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA, L-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue of L-AASA and is competitive vs AASA, uncompetitive vs NADPH, and noncompetitive vs L-glutamate. alpha-Ketoglutarate (alpha-Kg) serves as the dead-end analogue of L-glutamate and is competitive vs L-glutamate and uncompetitive vs L-AASA and NADPH. In the direction of saccharopine oxidation, N-oxalylglycine, L-pipecolic acid, L-leucine, alpha-ketoglutarate, glyoxylic acid, and L-ornithine were used as dead-end analogues of saccharopine and showed competitive inhibition vs saccharopine and uncompetitive inhibition vs NADP. The equilibrium constant for the reaction was measured at pH 7.0 by monitoring the change in absorbance of NADPH and is 200 M(-1). The value is in good agreement with the value determined using the Haldane relationship.     

Saccharopine dehydrogenase. Substrate inhibition studies.

In the direction of reductive condensation of alpha-ketoglutarate and lysine, saccharopine dehydrogenase (N6-(glutar-2-yl)-L-lysine:NAD oxidoreductase (lysine-forming) is inhibited by high concentrations of alpha-ketoglutarate and lysine, but not by NADH. NAD+ and saccharopine show no substrate inhibition in the reverse direction. Substrate inhibition by alpha-ketoglutarate and lysine is linear uncompetitive versus NADH. However, when the inhibition is examined with alpha-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, 301-307), the identity of values of inhibition constants of alpha-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+-alpha-ketoglutarate complex followed by the addition of lysine to this abortive complex.     

The reaction of pyruvate with saccharopine dehydrogenase.

The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxo-glutarate as a substrate of saccharopine dehydrogenase [epsilon-N-(L-glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine-forming) in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, L-lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2-oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+ was shown to be the last reactant released. Saccharopine [epsilon-N-(L-glutaryl-2)-L-lysine] was found to act as a potent dead-end inhibitor of the condensation reactions (of lysine and 2-oxoglutarate, and of lysine and pyruvate) by forming an abortive E. NADH. saccharopine complex.     

Kinetic mechanism of NAD:malic enzyme from Ascaris suum in the direction of reductive carboxylation

Initial velocity studies in the absence and presence of product and dead-end inhibitors suggest a steady-state random mechanism for malic enzyme in the direction of reductive carboxylation of pyruvate. For this quadreactant enzymatic reaction (Mn2+ is a pseudoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained at saturating concentrations while one reactant was varied at several fixed concentrations of the other. Data from the resulting reciprocal plots, analyzed in terms of a bireactant mechanism, are consistent with a sequential mechanism with an obligatory order of addition of metal prior to pyruvate. NAD is competitive against NADH whether pyruvate and CO2 are maintained at low or high concentrations, whereas it is noncompetitive against pyruvate and CO2. Thio-NADH, alpha-ketobutyrate, and nitrite were used as dead-end analogs of NADH, pyruvate, and CO2, respectively. Thio-NADH is competitive against NADH, whereas it is noncompetitive against pyruvate and CO2, in accordance with a random mechanism. alpha-Ketobutyrate and nitrite gave noncompetitive inhibition against all substrates. The noncompetitive patterns observed for alpha-ketobutyrate versus pyruvate and nitrite versus CO2 suggest binding of the inhibitor to both the E.Mn.NADH and E.Mn.NAD complexes. Primary deuterium isotope effects are equal on all kinetic parameters, in agreement with the random mechanism, and suggest equal off-rates for NAD from E.Mn.NAD as well as pyruvate and NADH from E.Mn.NADH.pyruvate. Data are consistent with an overall symmetry in the malic enzyme reaction in the two reaction directions with a requirement for metal bound prior to pyruvate and malate.     

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate.

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.     

Determinants of substrate specificity for saccharopine dehydrogenase from Saccharomyces cerevisiae

A survey of NADH, alpha-Kg, and lysine analogues has been undertaken in an attempt to define the substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substrates and dinucleotides important for substrate binding. A number of NAD analogues, including NADP, 3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), and thionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deamination reaction, as can a number of alpha-keto analogues, including glyoxylate, pyruvate, alpha-ketobutyrate, alpha-ketovalerate, alpha-ketomalonate, and alpha-ketoadipate. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with three carbons (from the alpha-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the alpha-keto acid is also important for binding; the alpha-oxo group contributes a factor of 10 to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic, and the optimal length of the hydrophobic portion of the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocket can accommodate a branch at the gamma-carbon, but not at the beta-carbon.     

Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid are mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphate ribose. NAD inhibits the IMPDH reaction at concentrations greater than 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (alpha) between 0 and 1. However, alpha was greater than 1 in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.     

Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae

The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product inhibition by OAS is noncompetitive against acetyl CoA, while it is uncompetitive against L-serine. Product inhibition by L-serine in the reverse reaction direction is noncompetitive with respect to both OAS and CoASH. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive versus L-serine and uncompetitive versus acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. Desulfo-CoA was used as a dead-end analog of both acetyl CoA and CoASH, and is competitive versus both substrates in the direction of L-serine acetylation; while it is competitive against CoASH and noncompetitive against OAS in the direction of CoASH acetylation. All of the above kinetic parameters are consistent with those predicted for an ordered mechanism at pH 6.5 with the exception of the uncompetitive inhibition by OAS vs. serine. The latter inhibition pattern suggests combination of OAS with the central E:acetyl CoA:serine complex. Cysteine is known to regulate its own biosynthesis at the level of SAT. As a dead-end inhibitor, L-cysteine is competitive against both substrates in both reaction directions. These results are discussed in terms of the mechanism of regulation.     

Kinetic mechanism of Ascaris suum phosphofructokinase desensitized to allosteric modulation by diethylpyrocarbonate modification

The kinetic mechanism of phosphofructokinase has been determined at pH 8 for native enzyme and pH 6.8 for an enzyme desensitized to allosteric modulation by diethylpyrocarbonate modification. In both cases, the mechanism is predominantly steady state ordered with MgATP binding first in the direction of fructose 6-phosphate (F6P) phosphorylation and rapid equilibrium random in the direction of MgADP phosphorylation. This is a unique kinetic mechanism for a phosphofructokinase. Product inhibition by MgADP is competitive versus MgATP and noncompetitive versus F6P while fructose 1,6-bisphosphate (FBP) is competitive versus fructose 6-phosphate and uncompetitive versus MgATP. The uncompetitive pattern obtained versus F6P is indicative of a dead-end E.MgATP.FBP complex. Fructose 6-phosphate is noncompetitive versus either FBP or MgADP. Dead-end inhibition by arabinose 5-phosphate or 2,5-anhydro-D-mannitol 6-phosphate is uncompetitive versus MgATP corroborating the ordered addition of MgATP prior to F6P. In the direction of MgADP phosphorylation, inhibition by anhydromannitol 1,6-bisphosphate is noncompetitive versus MgADP, while Mg-adenosine 5'(beta, gamma-methylene)triphosphate is noncompetitive versus FBP. Anhydromannitol 6-phosphate is a slow substrate, while anhydroglucitol 6-phosphate is not. This suggests that the enzyme exhibits beta-anomeric specificity.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae

Saccharomyces cerevisiae mitochondria contain an NADH:Q6 oxidoreductase (internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII. This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. From a structural point of view, the mature enzyme has a single subunit of 53 kDa with FAD as the only prosthetic group. Due to the fact that S. cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respiratory conditions. The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a Km for NADH of 9.4 microM and a Km for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 microM. NAD+, one of the products of the reaction, did not inhibit the enzyme while the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11.5 microM. Two dead-end inhibitors, AMP and flavone, were used to further characterize the kinetic mechanism of the enzyme. AMP was a linear competitive inhibitor of NADH (Ki = 5.5 mM) and a linear uncompetitive inhibitor of oxidized DCPIP (Ki = 11.5 mM), in agreement with the ping-pong mechanism. On the other hand, flavone was a partial inhibitor displaying a hyperbolic uncompetitive inhibition regarding NADH, and a hyperbolic noncompetitive inhibition with respect to oxidized DCPIP. The apparent intercept inhibition constant (Kii = 5.4 microM) and the slope inhibition constant (Kis = 7.1 microM) were obtained by non linear regression analysis. The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, although with lower efficiency. The effect of pH on Vmax was studied. The Vmax profile shows two groups with pKa values of 5.3 and 7.2 involved in the catalytic process.     

Enzymatic measurement of saccharopine with saccharopine dehydrogenase

We have developed an enzymatic method for measuring saccharopine, a key intermediate in lysine metabolism. With the enzyme saccharopine dehydrogenase, saccharopine can be oxidized to lysine and alpha-ketoglutarate with the corresponding conversion of NAD to NADH. The natural equilibrium favors saccharopine formation, but using hydrazine to trap one of the products, alpha-ketoglutarate, shifts the reaction toward quantitative oxidation of saccharopine. A stable endpoint is reached in 15-20 min, and although high concentrations of alpha-ketoglutarate slow the reaction, the end product is fully recovered. Unlike previous assays this technique is specific, convenient, and capable of measuring saccharopine directly in protein-free biological fluids or extracts.     

Complexes of NADH with betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus L

The kinetic mechanism of betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus is ordered with NAD(+) adding first. NADH is a noncompetitive inhibitor against NAD(+), which was interpreted before as evidence of an iso mechanism, in which NAD(+) and NADH binds to different forms of free enzyme. With the aim of testing the proposed kinetic mechanism, we have now investigated the ability of NADH to form different complexes with the enzyme. By initial velocity and equilibrium binding studies, we found that the steady-state levels of E.glycine betaine are negligible, ruling out binding of NADH to this complex. However, NADH readily bind to E.betaine aldehyde, whose levels most likely are kinetically significant given its low dissociation constant. Also, NADH combined with E.NADH and E.NAD(+). Finally, NADH was not able to revert the hydride transfer step, what suggest that there is no acyl-enzyme intermediate, i.e. the release of the reduced dinucleotide takes place after the deacylation step. Although formation of the complex E.NAD(+).NADH would produce an uncompetitive effect in the inhibition of NADH against NAD(+), the iso mechanism cannot be conclusively discarded.     

Kinetic study of porcine kidney betaine aldehyde dehydrogenase

Porcine kidney betaine aldehyde dehydrogenase (EC 1.2.1.8) kinetic properties were determined at low substrate concentrations. The double-reciprocal plots of initial velocity versus substrate concentration are linear and intersect at the left of the 1/v axis and showed substrate inhibition with betaine aldehyde. Studies of inhibition by NADH and dead-end analogs showed that NADH is a mixed inhibitor against NAD(+) and betaine aldehyde. AMP is competitive with respect to NAD(+) and mixed with betaine aldehyde. Choline is competitive against betaine aldehyde and uncompetitive with respect to NAD(+). The kinetic behavior is consistent with an Iso-Ordered Bi-Bi Steady-State mechanism.     

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Complete kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.     

Rhodococcus L-phenylalanine dehydrogenase: kinetics, mechanism, and structural basis for catalytic specificity

Phenylalanine dehydrogenase catalyzes the reversible, pyridine nucleotide-dependent oxidative deamination of L-phenylalanine to form phenylpyruvate and ammonia. We have characterized the steady-state kinetic behavior of the enzyme from Rhodococcus sp. M4 and determined the X-ray crystal structures of the recombinant enzyme in the complexes, E.NADH.L-phenylalanine and E.NAD(+). L-3-phenyllactate, to 1.25 and 1.4 A resolution, respectively. Initial velocity, product inhibition, and dead-end inhibition studies indicate the kinetic mechanism is ordered, with NAD(+) binding prior to phenylalanine and the products' being released in the order of ammonia, phenylpyruvate, and NADH. The enzyme shows no activity with NADPH or other 2'-phosphorylated pyridine nucleotides but has broad activity with NADH analogues. Our initial structural analyses of the E.NAD(+).phenylpyruvate and E.NAD(+). 3-phenylpropionate complexes established that Lys78 and Asp118 function as the catalytic residues in the active site [Vanhooke et al. (1999) Biochemistry 38, 2326-2339]. We have studied the ionization behavior of these residues in steady-state turnover and use these findings in conjunction with the structural data described both here and in our first report to modify our previously proposed mechanism for the enzymatic reaction. The structural characterizations also illuminate the mechanism of the redox specificity that precludes alpha-amino acid dehydrogenases from functioning as alpha-hydroxy acid dehydrogenases.     

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

Intra- and intermolecular electron transfer processes in redox proteins

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 ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-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.