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PubMedDate not supplied

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria.

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.

Chemical Precipitation↗
PubMed2001

Sodium pyruvate is better than sodium chloride as a resuscitation solution in a rodent model of profound hemorrhagic shock.

Pyruvate is an energy substrate that has both inotropic and antioxidant properties. In this study, we tested the hypothesis that survivorship would be better after resuscitation with 1.7% sodium pyruvate than 0.9% sodium chloride in a profound hemorrhagic shock model. The study was performed in a blinded manner. Rats were randomly assigned into two groups (ten in each group), a sodium chloride resuscitation group and a sodium pyruvate resuscitation group. After a 60-min shock period, we infused 80 ml/kg of a resuscitation solution. We continuously monitored mean arterial pressure and heart rate for 50 min after resuscitation. We recognized death by the disappearance of blood pressure pulsation and precordial movement. We performed a comparison of survivorship at 50 min post resuscitation using a Z-test of proportions. Nine (90%) of the animals that received sodium pyruvate were living 50 min after resuscitation, whereas only three (30%) of the animals that received sodium chloride survived to the same time point. We conclude that sodium pyruvate is better than sodium chloride as a resuscitation solution in a model of profound hemorrhagic shock.

Animals↗
PubMed1997

Monocarboxylates (pyruvate and lactate) as alternative energy substrates for the induction of long-term potentiation in rat hippocampal slices.

We previously reported that mild hypoglycemic conditions hamper the induction of long-term potentiation (LTP) in the CA1 region of rat hippocampal slices. In the present study, we examined whether monocarboxylates (pyruvate and lactate) permit LTP induction when glucose is unavailable. During glucose deprivation, pyruvate and lactate not only maintained basal synaptic transmission but also allowed LTP induction, with pyruvate being more effective than lactate. Additionally, LTP inhibition by 5 microM iodoacetate, a glycolytic inhibitor, was prevented by addition of pyruvate but not lactate. These results suggest that monocarboxylates can be affected alternative energy substrates for LTP induction when glucose is not available. However, lactate and pyruvate are not identical as energy sources for LTP induction.

Animals↗
PubMed1999

The inhibitory effects of lipoic compounds on mammalian pyruvate dehydrogenase complex and its catalytic components.

To examine the stereospecific effects of lipoic compounds on pyruvate metabolism, the effects of R-lipoic acid (R-LA), S-lipoic acid (S-LA) and 1,2-diselenolane-3-pentanoic acid (Se-LA) on the activities of the mammalian pyruvate dehydrogenase complex (PDC) and its catalytic components were investigated. Both S-LA and R-LA markedly inhibited PDC activity; whereas Se-LA displayed inhibition only at higher concentrations. Examination of the effects on the individual catalytic components indicated that Se-LA inhibited the pyruvate dehydrogenase component; whereas R-LA and S-LA inhibited the dihydrolipoamide acetyltransferase component. The three lipoic compounds lowered dihydrolipoamide dehydrogrenase (E3) activity in the forward reaction by about 30 to 45%. The kinetic data of E3 showed that both R-LA and Se-LA are used as substrates by E3 for the reverse reaction. Decarboxylation of [1-14C]pyruvate via PDC by cultured HepG2 cells was not affected by R-LA, but moderately decreased with S-LA and Se-LA. These findings indicate that (i) purified PDC and its catalytic components are affected by lipoic compounds based on their stereoselectivity; and (ii) the oxidation of pyruvate by intact HepG2 cells is not inhibited by R-LA. The later finding with the intact cells is in support of therapeutic role of R-LA as an antioxidant.

Acetyltransferases↗
PubMed2000

Fructose-mediated damage to lens alpha-crystallin: prevention by pyruvate.

Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that alpha-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free -NH(2) groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from alpha-crystallin was more pronounced than from beta-, and gamma-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.

Animals↗
PubMed2003

Septal infusions of glucose or pyruvate, but not fructose, produce avoidance deficits when co-infused with the GABA agonist muscimol.

Although glucose typically enhances memory or reverses memory deficits, glucose can also produce memory deficits when co-infused with the gamma-aminobutyric acid (GABA) agonist muscimol into the medial septum (Parent & Gold, 1997; Parent, Laurey, Wilkniss, & Gold, 1997). To date the mechanisms underlying the memory-impairing interaction between GABA and glucose remain unknown. Here we investigate whether this effect is the result of hyperosmolar conditions or may involve glucose metabolism. Male Sprague-Dawley rats were given one-trial inhibitory avoidance training after receiving septal infusions of vehicle (phosphate-buffered saline, 0.5 microl), the GABA(A) agonist muscimol (3 nmol), glucose (16.5, 33, or 66 nmol), fructose (33 nmol), pyruvate (33 nmol), or a solution containing muscimol combined with glucose, fructose, or pyruvate. Retention performance was tested 48 h later. Infusions of glucose, pyruvate, fructose, or muscimol alone did not affect retention performance. However, co-infusions of all doses of glucose (16.5, 33, or 66 nmol) or the glycolytic end product pyruvate with muscimol impaired retention performance. Co-infusions of fructose with muscimol did not affect retention performance. These results suggest that the memory-impairing interaction between glucose and muscimol does not result from hyperosmolar conditions, because equiosmolar concentrations of fructose do not mimic the effects of glucose and the memory deficits do not vary as a function of glucose concentration. The finding that pyruvate mimicked the effects of glucose and impaired memory when combined with muscimol suggests that glucose metabolism may be involved in the memory-impairing interaction between glucose and GABA(A) receptors in the medial septum.

Animals↗
PubMed1995

Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase. 2. Site-directed mutagenesis of an essential arginine contained within an apparent P-loop.

Pyruvate phosphate dikinase catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP). The Arg 561 residue of Clostridium symbiosum PPDK is contained within a Gly-rich stretch of sequence spanning positions 553-563 (viz., GAEGIGLCRTE) located in the 35 kDa C-terminal domain of the enzyme. The possible role of this stretch of sequence as a phosphate binding loop participating in catalysis of the PEP/pyruvate partial reaction (viz., E+PEP<-->E-P+pyruvate, where E-P represents enzyme phosphorylated at the catalytic histidine) was deduced from the similarity of this sequence to other known phosphate binding loops and by its location in the 35 kDa PEP/pyruvate binding domain of PPDK. To test the proposed role of Arg 561, and hence, the signature sequence, in catalysis of the E+PEP<-->E-P+pyruvate partial reaction, the C. symbiosum PPDK site-directed mutants Arg 561-->Leu 561 and Arg 561-->Lys 561 were constructed and expressed in Escherichia coli JM101. Neither mutant catalyzed the full PPDK reaction, ATP+P(i)+pyruvate<-->AMP+PP(i)+PEP, but both catalyzed the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction as efficiently as wild-type PPDK. Both mutants were shown to be unable to catalyze the PEP/pyruvate partial reaction. On the basis of these results it was proposed that Arg 561 and, possibly, the Gly-rich stretch of sequence spanning positions 553-563 are essential components of the active site of the PEP/pyruvate partial reaction.

Amino Acid Sequence↗
PubMed1982

Pyruvate kinase: is the mechanism of phospho transfer associative or dissociative?

To test for the possibility that pyruvate kinase proceeds via a dissociative path, we have investigated whether the complex enzyme . ADP . metaphosphate is transiently formed from the complex enzyme . ATP. It is shown that when highly purified pyruvate kinase is used, the rate of positional oxygen isotope exchange in ATP (beta, gamma bridge and beta nonbridge) is about 10(4) times slower in the absence of the cosubstrate pyruvate than it is in the presence of pyruvate. Further, the rate of racemization of the gamma-phospho group of [gamma (S)-16O,17O,18O]-ATP is undetectable, being at least 30 times slower even than the rate of positional isotope exchange. These tests thus provide no evidence that pyruvate kinase follows a dissociative mechanism. Indeed, it is argued that the available data are more consistent with an associative path. Evidence is presented that the single, associative, transition state is symmetrical, in which bond making and bond breaking processes are rather precisely balanced.

Adenosine Diphosphate↗
PubMed1985

pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 2. Physiological substrates and phosphoenol-alpha-ketobutyrate.

pH profiles have been determined for the reactions catalyzed by pyruvate kinase between pyruvate and MgATP and between phosphoenolpyruvate and MgADP. V, V/KMgATP, and V/Kpyruvate all decrease below a pK of 8.3 and above one of 9.2. The group with pK = 8.3 is probably a lysine that removes the proton from pyruvate during enolization, while the pK of 9.2 is that of water coordinated to enzyme-bound Mg2+. The fact that this pK shows in all three pH profiles shows that pyruvate forms a predominantly second sphere complex and cannot replace hydroxide to form the inner sphere complex that results in enolization and subsequent phosphorylation. On the basis of the displacement of the pK of the acid-base catalytic group in its V/K profile, phosphoenolpyruvate is a sticky substrate, reacting to give pyruvate approximately 5 times faster than it dissociates. The V/K profile for the slow substrate phosphoenol-alpha-ketobutyrate shows the pK of 8.3 for the acid-base catalytic group in its correct position, but this group must be protonated so that it can donate a proton to the intermediate enolate following phosphoryl transfer. The secondary phosphate pK of the substrate is seen in this V/K profile as well as in the pKi profile for phosphoglycolate (but not in those for glycolate O-sulfate or oxalate), showing a preference for the trianion for binding. The chemical mechanism with the natural substrates thus appears to involve phosphoryl transfer between MgADP and a Mg2+-bound enolate with metal coordination of the enolate serving to make it a good leaving group.

Adenosine Diphosphate↗
PubMed1989

Electron spin echo envelope modulation studies of pyruvate kinase active-site complexes.

Electron spin echo envelope modulation (ESEEM) spectroscopy, with Mn2+ and VO2+ as paramagnetic probes, was used to examine active-site structures at the protein-based divalent cation site of rabbit muscle pyruvate kinase in the presence of substrates, products, and the requisite inorganic cofactors. Two different VO.protein complexes were clearly distinguished, which differed with respect to coordination of the active-site lysine to VO2+. Lysine coordination was sensitive to the presence of pyruvate and phosphoenolpyruvate (PEP) and to the nature of the monovalent cation. In the presence of MgATP and oxalate, a 4-MHz 31P contact interaction was observed, which indicates that the ATP is directly coordinated to Mn2+ at the protein-based site. No 31P contact interactions were observed, however, in the presence of PEP. Pyruvate was determined to be a bidentate ligand of VO2+, on the basis of the observation of 2.2- and 5.4-MHz 13C contact interactions between VO2+ and [1-13C]pyruvate and [2-13C]pyruvate, respectively. Magnetic coupling between VO2+ or Mn2+ and 23Na, 39K, and 133Cs was observed, demonstrating the close proximity of the monovalent cation and the protein-based divalent cation.

Animals↗
PubMed1978

Mechanistic study of the addition of pyruvate to NAD+ catalyzed by lactate dehydrogenase.

The binary complex of NAD+ and dogfish A4 lactate dehydrogenase reacts reversibly with pyruvate enol to produce an inactive, enzyme-adduct complex, in which the nicotinamide and pyruvate moieties are linked by means of a covalent bond. This process is examined in both the forward and reverse directions as a function of reactant and buffer concentrations at pH 7, under conditions where the enolization of pyruvate is at equilibrium, and the involvement of complexes with stoichiometry E.NAD, E.NAD.PyrE, and E.NAD.PyrK is defined. (The subscripts, E and K, indicate the enol and keto forms of pyruvate.) One pathway for formation of the adduct complex involves the prior formation of the E.NAD.PyrE complex from E.NAD and pyruvate enol; the alternative pathway involves formation of the same complex via the enolization of E.NAD.PyrK, a process that is catalyzed by an external (nonenzymic) base. The possible use of the adduct reaction as a model for the normal enzymic reaction is considered.

Animals↗
PubMed1975

Pyruvate kinase isozymes in adult and fetal tissues of chicken.

Tissues of fetal and adult chickens were examined for pyruvate kinase activity. Two electrophoretically distinguishable and noninterconvertible isozymes were found. One of these, designated as type K (for kidney), is the sole pyruvate kinase in the early fetus and is found in appreciable quantities in all adult tissues except striated muscle. The second isozyme, type M, appears shortly before hatching in striated muscle and brain. These two isozymes correspond in their developmental pattern, tissue distribution, electrophoretic, immunological, and kinetic propertiesto similarly designated mammalian pyruvate kinases. However, no kinetic, immunological, or electrophoretic evidence could be found for a chicken isozyme corresponding to the mammalian type L pyruvate kinase. As the latter isozyme seems to be limited in its distribution mostly to highly differentiated gluconeogenic tissues (notable liver, kidney, and small intestine), our results support the proposition that the mammalian type L pyruvate kinase is a specilized isozyme that is present in mammals but not in birds.

Aging↗
PubMed1975

Properties of chicken skeletal muscle pyruvate kinase and a proposal for its evolutionary relationship to the other avian and mammalian isozymes.

Pyruvate kinase (EC 2.7.1.40) was isolated and purified from chicken and turkey breast muscle with a purification procedure very similar to that used for the bovine skeletal muscle isozyme (Cardenas, J., Dyson, R., and strandholm, J. (1973), J. Biol. Chem. 248,6931). A study of the chemical and physical properties of the chicken enzyme revealed that it is a tetramer of four apparently identical subunits, closely resembling in this and most other respects the mamalian type 7 isozyme. The properties of these two enzymes are similar enough to permit subunits of chicken type M pyruvate kinase to combine with subunits of mammalian type L (one of the three mammalian isozymes) to form interspecies tetrameric hybrid isozymes in relative quantities that do not differ makedly from those formed when both the M and L isozymes are of mammalian origin. The similarity between the mammalian and avian type M pyruvates kinases suggests a close evolutionary relationship. Further comparisons among the three mammalian and two avian isozymes of pyruvate kinase are consistent with a common evolutionary origin, perhaps from an ancestral form of the type K isozyme, which is the only pyruvate kinase identified in mammalian and avian embryos.

Amino Acids↗
PubMed1975

Magnetic resonance and kinetic studies of pyruvate, phosphate dikinase. Interaction of oxalate with the phosphorylated form of the enzyme.

Pyruvate, orthophosphate dikinase (EC 2.7.9.1) carries out its catalytic function in three successive partial reactions, the final step being the reaction of pyruvate with a stable phosphoenzyme intermediate to give phosphoenolpyruvate and free enzyme (Evans, H.J., and Wood, H. G. (1968), Proc. Natl. Acad. Sci. U.S.A. 61, 1448). Interactions of oxalate, a structural analog of enolpyruvate, with the phosphorylated form of the enzyme have been investigated by kinetic inhibition measurements and by magnetic resonance studies of manganous ion complexes with the enzyme. Oxalate inhibits the reaction catalyzed by pyruvate, phosphate dikinase, and the inhibition is linearly competitive with respect to pyruvate. The inhibitor constant for oxalate of 25 mu-M is fourfold lower than the Michaelis constant for pyruvate. The enhancement in the longitudinal relaxation rate of water protons (PRR) which occurs upon binding of Mn(II) to the enzyme has been used to monitor binding of oxalate to Mn(II)-enzyme complexes. PRR titrations indicate that the dissociation constant of oxalate from the Mn(II) complex of the free form of the enzyme is an order of magnitude weaker than the kinetically determined Ki. On the other hand, titrations of solutions which contain the phosphorylated form of the enzyme reveal a much stronger binding of oxalate. Moreover, the strength of oxalate binding to the phosphorylated enzyme is a function both of the species and of the concentration of monovalent cations in the solution. In the presence of Tl+, which has the most favorable activator constant for the final partial reaction, the dissociation constant for oxalate from its complex with the phosphorylated enzyme is less than 1 mu-M. Electron paramagnetic resonance (EPR) spectra for the enzyme-bound Mn(II) are sensitive to structural perturbations which occur upon binding of substrates or of oxalate to the enzyme. The EPR spectrum for the Mn(II)-phosphoenzyme-oxalate species is distinguished from spectra for other complexes of the enzyme by unusually narrow line widths and consequent resolution of fine structure from electronic quadrupole splitting. The narrow lines in the EPR spectrum are indicative of a rigid, pseudocrystalline environment for the bound Mn(II). The magnitude and frequency dependence of the PRR for the Mn(II)-phosphoenzyme-oxalate complex indicate that if any water molecules are bound to the Mn(II), their exchange with the bulk water is severely retarded. The kinetic and magnetic resonance studies support the hypothesis that oxalate mimics the reactive intermediate, enolpyruvate, in a complex with the phosphorylated enzyme which may resemble the structure of the transition state of the final partial reaction.

Bacillus↗
PubMed2003

(4-Hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis: the basis for ordered substrate addition.

(4-hydroxyphenyl)pyruvate dioxygenase (HPPD) catalyzes the second step in the pathway for the catabolism of tyrosine, the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate (HG). This reaction involves decarboxylation, substituent migration, and aromatic oxygenation. HPPD is a member of the alpha-keto acid dependent oxygenases that require Fe(II) and an alpha-keto acid substrate to oxygenate an organic molecule. We have examined the binding of ligands to HPPD from Streptomyces avermitilis. Our data show that HPP binds to the apoenzyme and that the apo-HPPD.HPP complex does not bind Fe(II) to generate active holoenzyme. The binding of HPP, phenylpyruvate (PPA), and pyruvate to the holoenzyme produces a weak ligand charge-transfer band at approximately 500 nm that is indicative of bidentate binding of the 1-carboxylate and 2-keto pyruvate oxygen atoms to the active site metal ion. For HPPD from this organism the 4-hydroxyl group of (4-hydroxyphenyl)pyruvate is a requirement for catalysis; no turnover is observed in the presence of phenylpyruvate. The rate constant for the dissociation of Fe(II) from the holoenzyme is 0.0006 s(-)(1) and indicates that this phenomenon is not significantly relevant in steady-state turnover. The addition of HPP and molecular oxygen to the holoenzyme is formally random. The basis of the ordered bi bi steady-state kinetic mechanism previously observed by Rundgren (Rundgren, M. (1977) J. Biol. Chem. 252, 5094-9) is the 3600-fold increase in oxygen reactivity when holo-HPPD is in complex with HPP. This complex reacts with molecular oxygen with a second-order rate constant of 1.4 x 10(5) M(-)(1) s(-)(1) inducing the formation of an intermediate that decays at the catalytically relevant rate of 7.8 s(-)(1).

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed2004

Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A.

The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.

Acetyl Coenzyme A↗
PubMed2006

EPR spectroscopic and computational characterization of the hydroxyethylidene-thiamine pyrophosphate radical intermediate of pyruvate:ferredoxin oxidoreductase.

The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at X-band and D-band microwave frequencies. EPR spectra, obtained with various combinations of isotopically labeled substrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations. Parameters obtained from the simulations were compared with those predicted from electronic structure calculations on various radical structures. The g-values and 14N/15N-hyperfine splittings obtained from the spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittings from the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP are consistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogen bond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar, sigma/n-type cation radical.

Electron Spin Resonance Spectroscopy↗
PubMed1998

Interfacial communications in recombinant rabbit kidney pyruvate kinase.

Tissue-specific isozymes of pyruvate kinase are particularly attractive systems to elucidate the molecular mechanism(s) of conferring allostery. The muscle- and kidney-type isozymes are coded by the same gene. As a consequence of alternative message RNA splicing, the two primary sequences differ by a small number of residues. However, they exhibit very different regulatory behavior. In an effort to identify the roles of specific residues in conferring allostery, the gene encoding rabbit kidney-type pyruvate kinase was cloned and expressed in Escherichia coli. The primary structure of recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, which are located in a region that forms important intersubunit contacts in the RMPK structure. Velocity sedimentation and analytical gel chromatographic studies show that rRKPK undergoes reversible dimer left and right arrow tetramer assembly with an equilibrium constant of 28 +/- 3 mL/mg. This subunit assembly process provides the opportunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effectors. The assembly to tetrameric rRKPK is favored by the binding of phosphoenolpyruvate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (FBP), an activator. In contrast, the equilibrium is shifted toward dimeric rRKPK upon binding of adenosine diphosphate (ADP), the other substrate, or l-phenylalanine (Phe), the inhibitor. These observations provide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system. First, all substrates and effectors communicate through this particular dimer-dimer interface. Second, the thermodynamic signatures of these communications are qualitatively different for the two substrates and between the activator, FBP, and inhibitor, Phe.

Amino Acid Sequence↗