Site-directed mutagenesis of a loop at the active site of E1 (α2β2) of the pyruvate dehydrogenase complex

2003 ◽  
Vol 270 (5) ◽  
pp. 861-870 ◽  
Author(s):  
Markus Fries ◽  
Hitesh J. Chauhan ◽  
Gonzalo J. Domingo ◽  
Hyo-Il Jung ◽  
Richard N. Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Pyruvate Dehydrogenase Complex ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Dehydrogenase Complex

scholarly journals Formation of N-hydroxy-N-arylacetamides from nitroso aromatic compounds by the mammalian pyruvate dehydrogenase complex

1993 ◽  
Vol 290 (3) ◽  
pp. 783-790 ◽  
Author(s):  
T Yoshioka ◽  
T Uematsu
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Aromatic Compounds ◽  
Pyruvate Dehydrogenase Complex ◽  
Catalytic Efficiency ◽  
Nitroso Compounds ◽  
Factors Affecting ◽  
Porcine Heart ◽  
Alkyl Groups ◽  
Dehydrogenase Complex

Bovine, human and porcine heart mitochondria and isolated porcine heart pyruvate dehydrogenase complex (PDHC) pyruvate-dependently form N-hydroxy-N-arylacetamides from nitroso aromatic compounds, including carcinogenic 4-biphenyl and 2-fluorenyl derivatives. The PDHC-catalysed formation of N-hydroxyacetanilide (N-OH-AA) from nitrosobenzene (NOB), through a Ping Pong mechanism, is optimum at pH 6.8 and is accelerated by thiamin pyrophosphate, but is inhibited by thiamin thiazolone pyrophosphate and ATP. Km pyruvate in the reaction is independent of pH over the range tested, whereas KmNOB increases at lower pH, owing to ionization of an active-site functional group of pKa 6.3. The enzymic ionization decreases log (Vmax/KmNOB). Isolated pyruvate dehydrogenase (E1), a constitutive enzyme of PDHC, forms N-OH-AA by itself and has comparable kinetic parameters to those of the PDHC-catalysed N-OH-AA formation. The catalytic efficiency of PDHC in the formation of N-hydroxy-N-arylacylamides, due to the steric limitation of the active site of E1, is lowered both by bulky alkyl groups of alpha-oxo acids and by p-substituents (but not an o-substituent) on nitrosobenzenes. These nitroso compounds serve as electrophiles in the reaction in which the reductive acetylation step is rate-limiting. The reaction mechanism and other factors affecting N-hydroxy-N-arylacylamide formation are discussed.

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

2000 ◽  
Vol 349 (1) ◽  
pp. 195-201 ◽  
Author(s):  
Jay J. THELEN ◽  
Jan A. MIERNYK ◽  
Douglas D. RANDALL
Keyword(s):  
Arabidopsis Thaliana ◽  
Pyruvate Dehydrogenase ◽  
Histidine Kinase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Primary Amino Acid Sequence ◽  
N Terminus ◽  
Primary Amino

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.

The pyruvate dehydrogenase multi-enzyme complex of Escherichia coli : genetic reconstruction and functional analysis of the lipoyl domains

1986 ◽  
Vol 317 (1540) ◽  
pp. 391-404 ◽  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Site Directed Mutagenesis ◽  
Enzyme Complex ◽  
Site Selective

The dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous lipoyl domains ( ca . 100 residues) that are tandemly repeated to form the N-terminal half of the polypeptide chain. These lipoyl domains are linked to a much larger ( ca . 300 residues) subunit-binding domain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. Selective in vitro deletions in the E2p gene ( aceF )have allowed the creation of truncated E2p chains in which one or more of the lipoyl domains has been excised. Site-directed mutagenesis has been used to change individual residues. The effects of these deletions and mutations on the assembly, catalytic activity and active-site coupling in the complex are assessed.

scholarly journals Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues. Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex

1990 ◽  
Vol 268 (1) ◽  
pp. 69-75 ◽  
Author(s):  
K P Williams ◽  
P F Leadlay ◽  
P N Lowe
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Trichomonas Vaginalis ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Decarboxylase ◽  
Oxidative Decarboxylation ◽  
Oxidoreductase Activity ◽  
Pyruvate:Ferredoxin Oxidoreductase ◽  
Thiamin Pyrophosphate ◽  
Dehydrogenase Complex

Pyruvate:ferredoxin oxidoreductase and the pyruvate dehydrogenase multi-enzyme complex both catalyse the CoA-dependent oxidative decarboxylation of pyruvate but differ in size, subunit composition and mechanism. Comparison of the pyruvate:ferredoxin oxidoreductase from the protozoon Trichomonas vaginalis and the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex shows that both are inactivated by incubation with pyruvate under aerobic conditions in the absence of co-substrates. However, only the former is irreversibly inhibited by incubation with hydroxypyruvate, and only the latter by incubation with bromopyruvate. Pyruvate:ferredoxin oxidoreductase activity is potently, but reversibly, inhibited by addition of bromopyruvate in the presence of CoA, and it is suggested that the mechanism involves formation of an adduct between CoA and bromopyruvate in the active site of the enzyme. It is proposed that both enzymes are inactivated by pyruvate through a mechanism involving oxidation of an enzyme-bound thiamin pyrophosphate/substrate adduct to form a tightly bound inhibitory species, possibly thiamin thiazolone pyrophosphate as hypothesized by Sumegi & Alkonyi.

scholarly journals A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

2007 ◽  
Vol 282 (38) ◽  
pp. 28106-28116 ◽  
Author(s):  
Sachin Kale ◽  
Palaniappa Arjunan ◽  
William Furey ◽  
Frank Jordan
Keyword(s):  
Escherichia Coli ◽  
Active Center ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Pyruvate Dehydrogenase Complex ◽  
Side Reactions ◽  
Thiamin Diphosphate ◽  
E Coli ◽  
Covalent Intermediate ◽  
Dehydrogenase Complex

Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.

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