Full Enzyme Complex Simulation: Interactions in Human Pyruvate Dehydrogenase Complex

2018 ◽  
Vol 58 (2) ◽  
pp. 362-369 ◽  
Author(s):  
Samira Hezaveh ◽  
An-Ping Zeng ◽  
Uwe Jandt
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Enzyme Complex ◽  
Complex Simulation ◽  
Dehydrogenase Complex

scholarly journals Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

1982 ◽  
Vol 205 (2) ◽  
pp. 389-396 ◽  
Author(s):  
Leonard C. Packman ◽  
Richard N. Perham ◽  
Gordon C. K. Roberts
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Random Coil ◽  
Cross Linking ◽  
Enzyme Complex ◽  
Cross Links ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the 1H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T2, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the 1H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by 1H n.m.r. spectroscopy.

scholarly journals Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides

1980 ◽  
Vol 189 (1) ◽  
pp. 161-172 ◽  
Author(s):  
C E Henderson ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Icosahedral Symmetry ◽  
Enzyme Complex ◽  
Molecular Masses ◽  
Dehydrogenase Complex

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.

Regulation of pyruvate dehydrogenase complex in ischemic rat heart

1984 ◽  
Vol 246 (6) ◽  
pp. H858-H864 ◽  
Author(s):  
T. B. Patel ◽  
M. S. Olson
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Total Activity ◽  
Control Flow ◽  
Enzyme Complex ◽  
Multienzyme Complex ◽  
Flow Rates ◽  
Perfused Rat Heart ◽  
Dehydrogenase Complex

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.

scholarly journals Inhibition of pyruvate dehydrogenase complex by moniliformin

1986 ◽  
Vol 233 (3) ◽  
pp. 719-723 ◽  
Author(s):  
P S Gathercole ◽  
P G Thiel ◽  
J H S Hofmeyr
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Inhibitory Action ◽  
Pyruvate Dehydrogenase Complex ◽  
Time Dependent ◽  
Enzyme Complex ◽  
First Order ◽  
Saturation Kinetics ◽  
Thiamin Pyrophosphate ◽  
Inhibition Reaction ◽  
Dehydrogenase Complex

The mechanism for the inhibition of pyruvate dehydrogenase complex from bovine heart by moniliformin was investigated. Thiamin pyrophosphate proved to be necessary for the inhibitory action of moniliformin. The inhibition reaction was shown to be time-dependent and to follow first-order and saturation kinetics. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin-inactivated complex only partially reversed inactivation. Moniliformin seems to act by inhibition of the pyruvate dehydrogenase component of the enzyme complex and not by acting on the dihydrolipoamide transacetylase or dehydrogenase components, as shown by monitoring the effect of moniliformin on each component individually. On the basis of these results, a suicide inactivator mechanism for moniliformin on pyruvate dehydrogenase is proposed.

scholarly journals Domain structure and1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus

1984 ◽  
Vol 217 (1) ◽  
pp. 219-227 ◽  
Author(s):  
L C Packman ◽  
R N Perham ◽  
G C K Roberts
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Bacillus Stearothermophilus ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Terminal Region ◽  
Enzyme Complex ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.

Isolierung und Charakterisierung eines membrangebundenen Pyruvatdehydrogenase-Komplexes aus dem phototrophen Bakterium Rhodospirillum rubrum /Isolation and Characterization of a Membrane-Bound Pyruvate Dehydrogenase Complex from the Phototrophic Bacterium Rhodospirillum rubrum

1977 ◽  
Vol 32 (5-6) ◽  
pp. 351-361 ◽  
Author(s):  
Rosemarie Lüderitz ◽  
Jobst-Heinrich Klemme
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Metal Ion ◽  
Rhodospirillum Rubrum ◽  
Coenzyme A ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Thiamine Pyrophosphate ◽  
Enzyme Complex ◽  
Isolation And Characterization ◽  
Dehydrogenase Complex

Abstract The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhododospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5 - 2 μmol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R . rubrum revealed no cooperativity between pyruvate binding sites. The kinetic constants [Km) for the overall reaction were (in mм) : 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The Km for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 μm . NADH was a potent inhibitor (Ki = 5 μm) of the enzyme complex and the dihydrolipo­ amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki = 40 μм) . The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mм) had no pronounced effect on the overall reaction.

scholarly journals Lipoic acid residues in a take-over mechanism for the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 513-520 ◽  
Author(s):  
J N Berman ◽  
G X Chen ◽  
G Hale ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Decarboxylase ◽  
Enzymic Activity ◽  
Enzyme Mechanism ◽  
Enzyme Complex ◽  
Complex Activity ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase complex of Escherichia coli contains two lipoic acid residues per dihydrolipoamide acetyltransferase chain, and these are known to engage in the part-reactions of the enzyme. The enzyme complex was treated with trypsin at pH 7.0, and a partly proteolysed complex was obtained that had lost almost 60% of its lipoic acid residues although it retained 80% of its pyruvate dehydrogenase-complex activity. When this complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, the rate of modification of the remaining S-acetyldihydrolipoic acid residues was approximately equal to the accompanying rate of loss of enzymic activity. This is in contrast with the native pyruvate dehydrogenase complex, where under the same conditions modification proceeds appreciably faster than the loss of enzymic activity. The native pyruvate dehydrogenase complex was also treated with lipoamidase prepared from Streptococcus faecalis. The release of lipoic acid from the complex followed zero-order kinetics for most of the reaction, whereas the accompanying loss of pyruvate dehydrogenase-complex activity lagged substantially behind. These results eliminate a model for the enzyme mechanism in which specifically one of the two lipoic acid residues on each dihydrolipoamide acetyltransferase chain is essential for the reaction. They are consistent with a model in which the dihydrolipoamide acetyltransferase component contains more lipoic acid residues than are required to serve the pyruvate decarboxylase subunits under conditions of saturating substrates, enabling the function of an excised or inactivated lipoic acid residue to be taken over by another one. Unusual structural properties of the enzyme complex might permit this novel feature of the enzyme mechanism.

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