Porcine Heart Mitochondrial Malate Dehydrogenase Research Articles

Crystallographic Studies of Glyoxysomal Malate Dehydrogenase Mutants D193N and H220Q

Crystallization was carried out by first dialyzing the purified enzyme against a buffer containing 10 mM NaH2PO4, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, pH 7.4. The dialyzed protein was concentrated to 10 mg/ml using an Amicon concentrator (PM10, 43 mm). Crystals of gMDH were grown at 20°C by hanging drop - vapor diffusion from a mother liquor containing 100 mM sodium citrate pH 4.5, 1 mM DTT, and PEG 8000. Needle-shaped crystals of gMDH appeared in a few days in the range of 6.5–11.0 % and 9.0–13.5% (w/v) of PEG 8000, respectively. gMDH was cryo-protected by adding a 50% glycerol solution to the crystallization drop resulting in a final concentration of 33% (v/v) glycerol. The crystal was then transferred to liquid nitrogen using a nylon loop. Data sets for native gMDH were collected at the SBC Beamline 19-ID (ANL-APS) with the 3x3 CCD area detector set at 2q = 0°. Phases were found for gMDH by molecular replacement using the dimeric structure of porcine heart mitochondrial malate dehydrogenase substituted with poly-alanine as a search model using X-PLOR. One dimer was present in the asymmetric unit. The phases of D157N gMDH were also found by molecular replacement using the partially refined model of gMDH and the program EPMR. Four dimers were present in the asymmetric unit of a P21 unit cell. Both x-ray structures were refined using Crystallography and NMR System (CNS v. 0.3). The models were refined by rigid body, then simulated annealing, positional refinement, and B-factor refinement. Five percent of the data were omitted from refinement and used for Rfree calculations. Non-crystallographic symmetry was used to restrain the 4 dimers of gMDH during the early refinement, then released during subsequent rounds. The program O was used for model rebuilding after each round of crystallographic refinement This work is supported by NSF Grant MCB 0448905 to EB

A "stripping" ligand tactic for use with the kinetic locking-on strategy: its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases.

The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27).

Substrate Channeling of NADH and Binding of Dehydrogenases to Complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.

Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase

Time-resolved fluorescence studies on the emission of NADH bound to porcine heart mitochondrial malate dehydrogenase (( S)-malate:NAD + oxidoreductase, EC 1.1.1.37), in the presence and absence of saturating levels of hydroxymalonate, were carried out. The lifetime of NADH bound in the ternary complex was determined to be 9.5 ns compared to 1.74 ns as reported in the literature. Steady-state and dynamic polarization data indicated a Debye rotational relaxation time in the range of 106–109 ns for the dimeric enzyme. This value is significantly larger than that calculated for a spherical protein and is consistent with the asymmetric dimer found by crystallographic studies.

The three-dimensional structure of porcine heart mitochondrial malate dehydrogenase at 3.0-A resolution.

In a previous study, we reported the apparent similarity between a low resolution electron density map of mitochondrial malate dehydrogenase and a model of cytoplasmic malate dehydrogenase (Roderick, S. L., and Banaszak, L. J. (1983) J. Biol. Chem. 258, 11636-11642). We have since determined the polypeptide chain conformation and coenzyme binding site of crystalline porcine heart mitochondrial malate dehydrogenase by x-ray diffraction methods. The crystals from which the diffraction data was obtained contain four subunits of the enzyme arranged as a "dimer of dimers," resulting in a crystalline tetramer which possesses 222 molecular symmetry. The overall polypeptide chain conformation of the enzyme, the location of the coenzyme binding site, and the preliminary location of several catalytically important residues have confirmed the structural similarity of mitochondrial malate dehydrogenase to cytoplasmic malate dehydrogenase and lactate dehydrogenase.

Regulation of mitochondrial malate dehydrogenase: Kinetic modulation independent of subunit interaction

Porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37), a dimeric enzyme of M r = 70,000, is both allosterically activated and inhibited by citrate. Using an affinity elution procedure based upon citrate binding to malate dehydrogenase, the isolation of pure heterodimer (a dimeric species with one active subunit and one iodoacetamide-inactivated subunit) has been achieved. Investigations utilizing this heterodimer in conjunction with resin-bound monomers of malate dehydrogenase have allowed the formulation of a definite conclusion concerning the role of subunit interactions in catalysis and regulation of this enzyme. The citrate kinetic effects, oxaloacetate inhibition, malate activation, and the effects of 2-thenoyl-trifluoroacetone (TTFA) are shown to be independent of interaction between catalytically active subunits. Previous kinetic data thought to support a reciprocating catalytic mechanism for this enzyme may be reinterpreted upon closer analysis in relation to an allosteric, conformationally specific binding model for malate dehydrogenase.

A facile method for the isolation of porcine heart mitochondrial malate dehydrogenase by affinity elution chromatography on Procion Red HE3B.

A quick, simple method has been devised for isolating pig heart mitochondrial malate dehydrogenase in apparently homogeneous state and good yield. It entails the adsorption of the enzyme to agarose-linked Procion Red HE3B and specific elution of a ternary complex consisting of the malate dehydrogenase, NAD+, and L-malate.

The conformation of mitochondrial malate dehydrogenase derived from an electron density map at 5.3-A resolution.

A monoclinic form of crystalline porcine heart mitochondrial malate dehydrogenase has been prepared and contains two dimers in the asymmetric unit. The analysis of single crystal x-ray data shows that the two dimers are packed in the crystal lattice as a tetramer with approximate 222-point symmetry. Heavy atom derivatives using the compounds ethylmercurithiosalicylic acid and IrCl3 have been characterized and multiple isomorphous replacement methods have been used to obtain an electron density map at 5.3-A resolution. The phasing of the x-ray data was aided by including contributions from crystal forms with and without the coenzyme, NAD+. By using electron density averaging methods, the quality of the low resolution electron density map was improved to the point where it was possible to establish the overall conformation of mitochondrial malate dehydrogenase. The results confirm the proposed similarities between the mitochondrial and cytoplasmic forms of the malate dehydrogenases. Studies using x-ray data from the apo- and holo-forms of the enzyme describe the location of the active site as well as the arrangement of subunits in the tetrameric crystalline form of the enzyme.

Regulation of mitochondrial malate dehydrogenase. Evidence for an allosteric citrate-binding site.

The effect of citrate on the structure and function of porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37) has been characterized. The native dimeric form of this enzyme is specifically activated by citrate in the NAD+ leads to NADH direction and inhibited by citrate in the NADH leads to NAD+ direction. It is proposed that citrate is bound at a regulatory site that is distinct from the catalytic site of the enzyme. In binding to this regulatory site, citrate greatly reduces the binding of NADH as determined by fluorescence titration and "Hummel-Dreyer"-type experiments, but does not diminish the binding of NAD+. As would be expected for an effector altering the equilibrium between two conformational forms of an enzyme, citrate favorably perturbs the equilibrium for the reaction in the direction of NAD+ reduction. Using [14C]citrate, the stoichiometry of citrate binding to mitochondrial malate dehydrogenase has been determined to be two equivalent sites per dimer, with a dissociation constant of 12.5 microM. In detailed kinetic studies, it has also been observed that activation by citrate abolishes (masks) the enzymatic activation induced by high concentrations of the substrate, L-malate. In addition, Hummel-Dreyer-type experiments indicate that less than a stoichiometric amount of NADH is bound to the enzyme under conditions of malate activation. These data are consistent with a previously suggested second "substrate" binding site proposed to explain the enzymatic activation observed at high concentrations of the substrate, L-malate (Telegdi, M., Wolfe, D. V., and Wolfe, R. G. (1973) J. Biol. Chem. 248, 6484-6489). This allosteric site may exist only on the enzyme conformation capable of binding NAD+.

Active subunits in hybrid-modified malate dehydrogenase.

Inactivation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by selective modification of an active center histidine residue with the reagent iodoacetamide has been further investigated to examine the existence of and the enzymatic activity of a hybrid (half)-modified dimer. The loss of enzymatic activity during iodo(1-14C) acetamide modification is linear with 14C incorporation. Enzyme was modified to various extents and the reaction was quenched. Microzonal electrophoresis was performed to separate native dimeric enzyme, hybrid-modified enzyme, and doubly modified enzyme. The distribution of each species was quantitated by scanning densitometry. The distribution generated throughout the time course of inactivation indicates that both subunits are modified independently and at the same rate. It is apparent that the hybrid-modified dimer contributes one-half of the enzymatic activity of a native dimer in the standard assay. Kinetic studies were performed and the results indicate that there is no apparent change in kinetic parameters between a subunit of the native dimer and the active subunit in the hybrid-modified dimer. Dissociation and reassociation of a mixture of native enzyme and doubly-iodoacetamide-modified enzyme indicates that there is no preferential association of a modified subunit with another modified subunit, or of a native subunit with another native subunit, but rather, association is random with respect to native and iodoacetamide-modified subunits.

The N-ethylmaleimide-sensitive cysteine residue in the pH-dependent subunit interactions of malate dehydrogenase.

The specific chemical modification by N-ethylmaleimide of a cysteine residue at pH 5.0 in porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been shown to result in an enzymatically inactive, monomeric product, which does not reassociate at pH 7.5 to yield the native dimer. In this report, an investigation of proton release and uptake upon NADH binding to the native enzyme and to the N-ethylmaleimide-modified enzyme has implicated the above cysteine residue as being directly linked to the pH-dependent subunit dissociation of mitochondrial malate dehydrogenase. The results are consistent with the view that the modified cysteine residue is not located at the subunit interaction site, although it is probably near this site. A recent study from this laboratory has demonstrated that the monomeric enzyme obtained at pH 5.0 exists in a conformation which is enzymatically inactive and which has an enhanced intrinsic protein fluorescence. Interpretation of protein fluorescence data has suggested that the N-ethylmaleimide modification results in inactivation of the enzyme by preventing the pH-induced conformational change to the active dimer. However, NADH is able to induce reassociation of the N-ethylmaleimide-modified enzyme at pH 7.5 but not at pH 5.0. This reassociation at pH 7.5 is accompanied by a significant regain of enzymatic activity, indicating that NADH binding is able to partially overcome the negative effect of the cysteine modification on the pH-dependent subunit reassociation of mitochondrial malate dehydrogenase.

The immobilization of mitochondrial malate dehydrogenase on Sepharose beads and the demonstration of catalytically active subunits.

Porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been immobilized by covalent attachment to CNBr-activated Sepharose 4B-Cl gel. The gel was activated with low levels of CNBr to produce a low density of linkage sites and, hence, to facilitate linkage of the enzyme through a single subunit. Matrix-bound mitochondrial malate dehydrogenase was found to possess 50-65% of the native mitochondrial malate dehydrogenase specific activity when assayed in the NAD+ leads to NADH direction but only 5-15% of the native enzyme specific activity when assayed in the NADH leads to NAD+ direction. MB-dimeric mitochondrial malate dehydrogenase was dissociated to MB-monomer by exposure to pH 5.0 buffer. The MB-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to MB-dimer. The reconstitution of Mb-monomer to MB-dimer was accomplished by adding dissociated mitochondrial malate dehydrogenase, which exists at pH 5.0, to MB-monomer and adjusting to pH 7.5. The kinetic parameters, pH activity profile, and stability toward heat denaturation for MB-mitochondrial malate dehydrogenase (monomer and dimer) were determined and compared to native mitochondrial malate dehydrogenase. MB-mitochondrial malate dehydrogenase exhibited enhanced stability and similar pH activity profiles when compared to native mitochondrial malate dehydrogenase. Immobilization of mitochondrial malate dehydrogenase altered the enzyme's kinetic parameters in such a manner as to increase the values of Km for the substrates and decrease the values of Vmax.

Subunit interactions in mitochondrial malate dehydrogenase. Kinetics and mechanism of reassociation.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been more extensively characterized. The native, dimeric form of the enzyme (Mr = 70,000) which exists at pH 7.5 has previously been shown to dissociate into its constituent subunits (Mr = 35,000) at pH 5.0 (Bleile, D. M., Schulz, R. A., Gregory, E. M., and Harrison, J. H. (1977) J. Biol. Chem. 252, 755-758). The dissociation is accompanied by a concomitant decrease in enzymatic specific activity and an increase in intrinsic protein fluorescence. By using the characteristics of specific activity and intrinsic protein fluorescence as probes of dimerization, the kinetics of subunit reassociation was investigated. In order to facilitate reassociation, a pH jump method was utilized in which enzyme at pH 5.0 was diluted into a large excess of pH 7.5 buffer. The regain of enzymatic specific activity and the decrease in protein fluorescence were observed to follow first order kinetics. The rate constant in both cases was dependent upon the protein concentration, and in all cases, full recovery of either enzymatic activity or native protein fluorescence was obtained. The Arrhenius activation energy for the reassociation of the subunits was found to be approximately 20 kcal/mol, an observation which is consistent with a refolding process whose rate-limiting step may be the cis/trans-isomerization about one or more proline imino bonds. A model for subunit reassociation which is consistent with the kinetic data is proposed.

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase ( l-malate: NAD + oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide ( E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta 253, 489–497 ) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. ( A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J. 173, 597–605 ) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem. 253, 8702–8707 ).

Investigation of the anticooperative binding of NADH to porcine heart mitochondrial malate dehydrogenase.

Investigation of the anticooperative binding of NADH to porcine heart mitochondrial malate dehydrogenase.

The relation of the pH and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase

The relationship of the pH-dependent and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) was investigated by means of gel filtration chromatography utilizing a standardized Sephacryl S-200 column. The results obtained indicate that the dimeric form of this enzyme dissociates to yield monomers at conditions of low protein concentration or at pH values below neutrality. In addition it is apparent that as the pH is lowered, the minimum concentration of protein required to maintain the enzyme in the dimeric form is increased.

Investigation of the relation of the pH-dependent dissociation of malate dehydrogenase to modification of the enzyme by N-ethylmaleimide.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been further characterized using the technique of sedimentation velocity ultracentrifugation. The increased rate and specificity of the inactivation of mitochondrial malate dehydrogenase by the sulfhydryl reagent N-ethylmaleimide has been correlated with the pH-dependent dissociation of the enzyme. Data obtained using NAD+ and its component parts to reassociate the enzyme and also to protect the enzyme from inactivation by N-ethylmaleimide suggest that the sulfhydryl residues being modified by N-ethylmaleimide are inaccessible when the enzyme is in its dimeric form. A dissociation curve for the pH-dependent dissociation suggests that a limited number of residues are being protonated concomitant with dissociation of the enzyme. An apparent pKa of 5.3 has been determined for this phenomenon. Studies using enzyme modified by the sulfhydryl reagent N-ethylmaleimide indicate that selective modification of essential sulfhydryl residues alters the proper binding of NADH.

Activation of porcine heart mitochondrial malate dehydrogenase by zero valence sulfur and rhodanese

Incubation of malate dehydrogenase with thiosulfate and rhodanese lead to an increase of dehydrogenasic activity. Selenosulfate, elemental sulfur and elemental selenium were shown similarly able to activate this protein. The activation is limited to the presence of SH groups on the protein. Experiments with 35S demonstrated the direct transfer of zero valence sulfur from rhodanese to malate dehydrogenase. It is proposed that this activation could be a mechanism of enzyme activity modulation in vivo.

Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.

Lipoic acid inhibition of mitochondrial malate dehydrogenase

Porcine heart mitochondrial malate dehydrogenase (L-malate:NAD + oxidoreductase, EC 1.1.1.37) has been shown to be inhibited by extremely low concentrations of lipoic acid. The actual inhibitor was found to be a high molecular weight substance, which can be separated by gel permeation from the non-inhibitory monomeric form of lipoic acid. This inhibitor has been identified as a polymeric form of lipoic acid.