Pyruvate Dehydrogenase Multienzyme Complex Research Articles

Substrate-induced structural changes of the pyruvate dehydrogenase multienzyme complex

The time course of the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex produces an unexpectedly high lag (τ = 8 s) even in the presence of saturating concentrations of its substrates. The preincubation of the pyruvate dehydrogenase complex with one of the substrates alone decreases the duration of this lag, and all the substrates of the pyruvate dehydrogenase component (E 1) and dihydrolipoyl transacetylase component (E 2) together (pyruvate, thiamine pyrophosphate, and CoA) result in the complete disappearance of the lag. The reduction of the dihydrolipoyl dehydrogenase component (E 3) of the pyruvate dehydrogenase complex with the substrates of the complex in the absence of NAD + produces significantly different quenching in the FAD fluorescence, and then the reduction with the substrates of E 3 as dihydrolipoic acid and dithioerythritol. (The formation of FADH 2 was not observed in the system.) The higher fluorescence quenching in the presence of substrates of pyruvate dehydrogenase complex compared to the effect caused by the substrates of the E 3 component (dihydrolipoic acid and DTE) indicates conformational changes additionally manifested in the fluorescence properties of the enzyme complex. The substrate-induced quenching of the enzyme-bound FAD fluorescence shows biphasic kinetics. The rate constant of the slow phase is comparable with the rate constant calculated from the time duration of the lag phase observed in the overall reaction. The kinetic analysis of both intensity and anisotropy decrease of the FAD fluorescence suggests a consecutive transmittance of an all substrate-coordinated, induced conformational changes directed from the pyruvate dehydrogenase-via the lipoyl transacetylase—to the lipoyl dehydrogenase. Two simultaneous conformational effects caused by binding of the substrates can be distinguished; one of them results the fluorescence of the bound FAD to be more quenched, while the other makes the FAD more mobile. The first-order rate constants of both these conformational changes were determined. The present observations suggest that the pyruvate dehydrogenase complex exists in a partially inactive state in the absence of its substrates, and it becomes active due to conformational changes caused by the binding of its substrates.

Regulation of Mammalian Pyruvate and Branched-Chain α-Keto Acid Dehydrogenase Complexes by Phosphorylation — Dephosphorylation

Publisher Summary The pyruvate dehydrogenase multienzyme complex and the branched-chain α-keto acid dehydrogenase multienzyme complex are located in mitochondria, within the inner membrane matrix compartment. The pyruvate dehydrogenase multienzyme complex and the branched-chain α-keto acid dehydrogenase multienzyme complex are located in mitochondria within the inner membrane matrix compartment. The pyruvate dehydrogenase complex is well designed for fine regulation of its activity. Interconversion of the active and inactive forms of pyruvate dehydrogenase is a dynamic process that leads rapidly to the establishment of steady states, in which the fraction of phosphorylated enzyme can be varied progressively over a wide range by changing the concentration or molar ratios of effectors that regulate activities of the kinase and the phosphatase. The pyruvate dehydrogenase complex, like other interconvertible enzyme systems, functions uniquely as a metabolic integration system. By means of multisite interactions with allosteric effectors, the kinase and the phosphatase can sense simultaneous fluctuations in the intracellular concentrations of several metabolites and adjust the specific activity of pyruvate dehydrogenase accordingly. This chapter illustrates the phosphorylation and concomitant inactivation of the α-ketoisovalerate dehydrogenase complex by its endogenous kinase, and the dephosphorylation and reactivation by the addition of highly purified α-ketoisovalerate dehydrogenase phosphatase.

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Bacillus subtilis.

Limited digestion of the pyruvate dehydrogenase complex of Bacillus subtilis with either trypsin or chymotrypsin at 0 degrees C inhibited its ability to decarboxylate pyruvate and 2-oxoisovalerate oxidatively, without causing disassembly of the complex. The proteinases selectively cleaved the E1 alpha subunits to form two fragments of Mr 31500 and approx. 9500, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both fragments remaining bound to the complex. Trypsin also caused a much slower cleavage of the E2 subunits, to form a fragment of apparent Mr 34000. The inhibition of overall dehydrogenase-complex activity was accompanied by the apparent loss of the pyruvate-driven and 2-oxoisovalerate-driven E1 activities, which was found to be due to a large increase in the Km for the 2-oxo acids: this change was correlated with the cleavage of the E1 alpha subunit.

Stabilizing effect of dichloroacetate on the pyruvate dehydrogenase-multienzyme complex

Stabilizing effect of dichloroacetate on the pyruvate dehydrogenase-multienzyme complex

N-(2,3-dimercaptopropyl)phthalamidic acid: Protection, in vivo and in vitro, against arsenic intoxication

The ip LD50s of N-(2,3-dimercaptopropyl)phthalamidic acid (DMPA) and British Anti-Lewisite (BAL) were 0.819 and 1.48 mmol/kg, respectively, in male albino mice. The ip ED50 of DMPA and BAL for prevention of the lethal effects of 0.15 mmol NaAsO 2/kg was 0.022 and 0.169 mmol/kg, respectively. DMPA increased the LD50 of sodium arsenite by approximately 2.5-fold following two ip injections of 0.20 mmol DMPA/kg. The effectiveness of DMPA in reducing the toxicity of NaAsO 2 was further demonstrated by its reversal of the sodium arsenite inhibition of pyruvate dehydrogenase multienzyme complex (PDH) activity in vitro. Similarly, in an in vivo experiment in which mice received 0.10 mmol NaAsO 2/kg, and 30 min later were given 0.05 or 0.10 mmol/kg DMPA, there was a rapid recovery of PDH activity. The distribution of 74As in the tissues of male New Zealand rabbits was altered following im injection of 0.20 mmol/kg DMPA. Under these conditions, the tissue concentration of 74As was significantly decreased. For all tissues tested, the 74As content decreased by at least 50% as compared to that of untreated controls. DMPA was effective also in increasing both urinary and fecal excretion of arsenic. The stability of aqueous solutions of DMPA varies with the pH of the solution. DMPA is more stable in acid solution.

Activation of pyruvate dehydrogenase complex (PDC) of rat heart mitochondria by glyburide

The effects of the second generation sulfonylurea, glyburide, on the pyruvate dehydrogenase multienzyme complex (PDC) of rat myocardial tissue were examined using rat ventricular slices and isolated mitochondria. Therapeutic concentrations (10 −7 to 10 −6M) of glyburide produced a 30% increase in the decarboxylation of [1 −14C] pyruvate by the PDC of ventricular tissue. Addition of glyburide to intact rat heart mitochondria stimulated activity of the PDC in a time- and concentration-dependent manner. Half-maximal stimulation of the enzyme occurred with 6 × 10 −5M glyburide and maximal activation of the enzyme was achieved with 1 × 10 −4M glyburide. At the height of stimulation, PDC activities were 6-fold greater than those observed under control conditions with succinate alone. When mitochondria were disrupted by sonication or freezethawing, glyburide produced no stimulation of pyruvate decarboxylation. We conclude that glyburide directly stimulates the decarboxylation of pyruvate by the PDC of the myocardium. Furthermore, the presence of intact mitochondria is necessary for the stimulatory action of glyburide on the PDC.

Spin-label studies on the lipoamide residues of the pyruvate dehydrogenase multienzyme complex of escherichia coli

The catalytic mechanism of the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been investigated using spin-labelling and synthetic inhibitors of the enzyme. The lipoamide residues covalently bound to the lipote acetyltransferase component of the complex were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl in the presence of pyruvate. The electron spin resonance spectrum of the spin-labelled enzyme displayed two components, one highly mobile (correlation time about 1 ns) and the other immobilized (correlation time much greater than 50 ns). The temperature-dependence of the signal intensities of the two components suggested that the lipoamide residues can occupy two alternative states, the proportion of spin-labeled lipaomide in the mobile state increasing as the temperature is raised. Pyruvate caused a fall in the intensity of the ESR signal from the spin-labelled lipaomide residues in the presence of thiamine pyrophosphate and Mg 2+. Acetoin also led to loss of ESR signal, but, with both pyruvate and acetoin, the additio of thiamine thiazolone pyprophosphate prevented the loss of signal. The rate of loss of ESR signal intensity was consistent with an intramolecular reaction between lipaomide-bound nitroxide groups and hydroxyethylthiamine pyrophosphate. The similar rates of reduction by isoascorbate of nitroxide spin-lebel free in solution and bound to the lipoamide residues of the pyruvate dehydrogenase complex indicated free access of the isoascorbate to the enzyme-boynd nitroxide groups. 13P-NMR of acetyl phosphonate, a competitive inhibitor of the enzyme, bound to native enzyme and to spin-labelled enzyme, showed a large chemical shift on binding, but the linewidths did not indicate any spin-spin interaction between the nitroxide radicals and the 31P nuclei. These results agree with recent experimental data from kinetic studies and 1H-NMR spectroscopy on the system.

Activation of pyruvate dehydrogenase complex by porcine and biosynthetic human insulin in cultured human fibroblasts.

Cultured human fibroblasts represent an appropriate model for studying both insulin receptor interaction and hormone responsiveness. We have investigated the properties of the pyruvate dehydrogenase multi-enzyme complex (PDC) and have studied the effects of various concentrations of porcine and biosynthetic human insulin (BHI) on the activity of the enzyme. Under optimal conditions of the assay, both BHI and porcine insulin activated PDC in a dose-dependent fashion in which full activation of the enzyme was achieved with 10(-8) M insulin. The half-maximal concentration for porcine and human insulin was similar, occurring at the level of 5 X 10(-9) M for activation of the PDC of human fibroblasts. We conclude that the PDC of cultured human fibroblasts is activated by both human and porcine insulin at a comparable physiologic concentration. Human fibroblasts may therefore serve as a useful model to study insulin action in isolated human tissue.

Regulation of pyruvate dehydrogenase complex in ischemic rat heart.

The effect of flow-induced ischemia on the rate of pyruvate decarboxylation and the activation state of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated, perfused rat heart. Pyruvate dehydrogenase activity in the heart decreased significantly during flow-induced ischemia and was a function of changes in the activation state (i.e., active/total activity) of the enzyme complex. In the absence of pyruvate, the activation state of pyruvate dehydrogenase decreased from nearly 100% active at the normal flow rate (10 ml/min) to 20% active as the flow was reduced to 0.5 ml/min. At high pyruvate levels (5 mM), the activation state increased from nearly 70% active at control flow rates to 100% active during ischemia. At an intermediate pyruvate concentration (0.5 mM), the enzyme complex was maintained at a relatively low activation state (30-35% active) throughout the range of flow rates tested. Ischemia caused elevated perfusate lactate concentrations only when the flow rates were less than 5.0 ml/min. The activation state of the pyruvate dehydrogenase complex in hearts perfused with glucose was also decreased during ischemia.

Repeating functional domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Each polypeptide chain in the lipoate acetyltransferase (E2) core of the pyruvate dehydrogenase complex from Escherichia coli contains three repeating sequences in the N-terminal half of the molecule. The repeats are highly homologous in primary structure and each includes a lysine residue that is a potential site for lipoylation. We have shown that all three sites are lipoylated, at least in part, and that the three lipoylated segments of the E2 chain can be isolated as distinct functional domains after limited proteolysis. Each domain becomes partly acetylated in the intact complex in the presence of substrate. In the primary structure, the domains are separated by regions of polypeptide chain oddly rich in alanine and proline residues. These regions are probably the conformationally mobile segments observed in the 1H-n.m.r. spectrum of the complex and which are removed by tryptic cleavage at Lys-316. The C-terminal half of the molecule contains the acetyltransferase active site and the binding sites for E1, E3 and other E2 subunits. The pyruvate dehydrogenase complex of E. coli, which has a heterogeneous quaternary structure, is thus far unique among the 2-oxo acid dehydrogenase complexes in possessing more than one lipoyl domain per E2 chain, but this may be a general feature of the enzyme from Gram-negative organisms.

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for an essential histidine residue at the active site of lipoamide dehydrogenase.

Incubation of pyruvate dehydrogenase multienzyme complex (PD complex) from Escherichia coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity. The mode of inactivation occurred by initial "anchoring" of the reagent via its -AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189]. To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates. E3 subunits were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea. Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis. The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine. These residues were not present in significant amounts in the controls. The loss of 81% of E3 activity correlated with the alkylation of about 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3.

Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Bromopyruvate behaves as an active-site-directed inhibitor of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex of Escherichia coli. It requires the cofactor thiamin pyrophosphate (TPP) and acts initially as an inhibitor competitive with pyruvate (Ki ca. 90 microM) but then proceeds to react irreversibly with the enzyme, probably with the thiol group of a cysteine residue. E1 catalyzes the decomposition of bromopyruvate, the enzyme becoming inactivated once every 40-60 turnovers. Bromopyruvate also inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different. Under these conditions, bromopyruvate is decarboxylated, and the lipoic acid residues in the lipoate acetyltransferase (E2) component become reductively bromoacetylated. Further bromopyruvate then reacts with the new thiol groups thus generated in the lipoic acid residues, inactivating the complex. If reaction with the lipoic acid residues is prevented by prior treatment of the complex with N-ethylmaleimide in the presence of pyruvate, the mode of inhibition reverts to irreversible reaction with the E1 component. In both types of inhibition of E1, reaction of 1 mol of bromopyruvate/mol of E1 chain is required for complete inactivation, and all the evidence is consistent with reaction taking place at or near the pyruvate binding site.

Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes.

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes of Escherichia coli, ox heart and Bacillus stearothermophilus was measured at various temperatures. As the temperature was raised, the extent of active-site coupling was found to increase, approaching a maximum near the physiological growth temperature of the organism. Under these conditions, a single pyruvate dehydrogenase (lipoamide) dimer appeared able to cause a rapid (20s) reductive acetylation of probably all 24 polypeptide chains in the dihydrolipoamide acetyltransferase core of the enzyme complex from E. coli at 37 degrees C, and of most if not all of the 60 polypeptide chains in the dihydrolipoamide acetyltransferase cores of the enzymes from ox heart and B. stearothermophilus at 37 degrees C and 60 degrees C respectively. Experiments designed to measure the inter-core and intra-core migration of enzyme subunits suggested that, in the bacterial enzymes at least, this was not a major contributor to active-site coupling.

A computer model analysis of the active-site coupling mechanism in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

A computer modeling system developed to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) accompanying release of lipoyl moieties by lipoamidase and by trypsin [Hackert, M.L., Oliver, R.M. & Reed, L.J. (1983) Proc. Natl. Acad. Sci. USA 80, 2226-2230] was used to analyze analogous data for the E. coli pyruvate dehydrogenase complex (PDC). The model studies indicate that the activity of PDC, as found for KGDC, is influenced by redundancies and random processes, which we describe as a multiple random coupling mechanism. In both complexes more than one lipoyl moiety services each pyruvate dehydrogenase (EC 1.2.4.1) or alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1) subunit, and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data for PDC was obtained with a model that has four lipoyl domains with four or, more probably, eight lipoyl moieties servicing each E1 subunit. The lipoyl-lipoyl interaction network for PDC has lipoyl domain interactions similar to those found for KGDC plus the additional possibility of interaction of a lipoyl moiety and its paired mate on each dihydrolipoamide acetyltransferase (EC 2.3.1.12) (E2) subunit. The two lipoyl moieties on an E2 subunit in PDC appear to be functionally indistinguishable, each servicing the acetyltransferase site of that E2 subunit and a dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3) subunit if the latter is bound to that particular E2 subunit. The observed difference between inactivation of PDC by lipoamidase and by trypsin appears to be due to dead-end competitive inhibition by lipoyl domains that have been modified by excision of lipoyl moieties by lipoamidase.

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP. It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring. The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin. Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli. The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated. The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP. Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP. The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein. A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.

Stabilized bacteria-based potentiometric electrode for pyruvate

A bacteria-based potentiometric electrode is described which demostrates a strategy for adapting an unstable enzyme system for use as a practical biocatalyst. Streptococcus faecium (ATCC 9790), containing the pyruvate dehydrogenase multienzyme complex, serves to convert pyruvate to carbon dioxide, which is detected by a gas-sensing electrode. The probe has an average slope of 41 m V per concentration decade over a range from 2.2 × 10 −4 to 3.2 × 10 −2 M pyruvate, and a lowe limit of detection of 1.3 × 10 −4 M pyruvate. Response times varied from 6–9 min for a new electrode to a maximum of 8—12 min during the probe lifetime of two weeks. Electode storage at 4°C in pH 5.5 buffer containing phosphate anions was critical optimal probe response characteristics. The Streptococcu faecium electrode is compared and contrasted with other potentiometric pyruvate electrodes reported employing isolated enzyme, dual enzyme—cofactor, or plant tissue biocatalysts.

Quaternary structure of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus studied by a new reversible cross-linking procedure with bis(imidoesters).

The bis(amidine) cross-links formed between protein subunits by treating them with bis(imidoesters) were found to be rapidly broken by exposing the cross-linked proteins to methylamine buffers containing the aprotic solvent acetonitrile. This cleavage step could be introduced between the two dimensions of a diagonal gel electrophoretic separation of cross-linked proteins to facilitate identification of the contributors to a cross-linked species. Tests with the tetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase demonstrated the simplicity and effectiveness of the technique. When the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was treated with a range of bis(imidoesters), from dimethyl succinimidate to dimethyl suberimidate, the most informative set of products was obtained with dimethyl glutarimidate. The longer bis(imidoesters) caused too extensive cross-linking of the enzyme subunits, although the beta chain of the pyruvate decarboxylase component always appeared to be the most resistant. Almost all the cross-linked species up to pentamers of the lipoate acetyltransferase polypeptide chain (apparent Mr approximately 280 000) were identified by means of the diagonal gel electrophoretic procedure after cleavage of the cross-links. The introduction of the methylamine cleavage step enables the bis(imidoester) for such experiments to be selected purely for the efficacity of its cross-linking reaction with the protein and dispenses with the need to incorporate a specially cleavable bond in the reagent.

Reconstitution of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTReconstitution of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilusRainer Jaenicke and Richard N. PerhamCite this: Biochemistry 1982, 21, 14, 3378–3385Publication Date (Print):July 1, 1982Publication History Published online1 May 2002Published inissue 1 July 1982https://doi.org/10.1021/bi00257a020RIGHTS & PERMISSIONSArticle Views67Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alerts Get e-Alerts

Limited proteolysis and proton NMR spectroscopy of Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.