Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Biochemistry ◽  
1985 ◽  
Vol 24 (25) ◽  
pp. 7187-7191 ◽  
Author(s):  
Larry R. Stepp ◽  
Lester J. Reed
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site

Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component fromEscherichia coliobserved at 2.32 Å resolution

2006 ◽  
Vol 62 (11) ◽  
pp. 1382-1386 ◽  
Author(s):  
Krishnamoorthy Chandrasekhar ◽  
Palaniappa Arjunan ◽  
Martin Sax ◽  
Natalia Nemeria ◽  
Frank Jordan ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Multienzyme 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.

Arginine-239 in the beta subunit is at or near the active site of bovine pyruvate dehydrogenase

1995 ◽  
Vol 1252 (2) ◽  
pp. 203-208 ◽  
Author(s):  
Devayani Eswaran ◽  
M. Showkat Ali ◽  
Bhami C. Shenoy ◽  
Lioubov G. Korotchkina ◽  
Thomas E. Roche ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Beta Subunit

scholarly journals Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

2011 ◽  
Vol 2011 ◽  
pp. 1-13
Author(s):  
Tripty A. Hirani ◽  
Alejandro Tovar-Méndez ◽  
Ján A. Miernyk ◽  
Douglas D. Randall
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Loop Structure ◽  
Hybrid Analysis ◽  
Mutant Proteins ◽  
Variable Substrate ◽  
Two Hybrid

We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana α2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.

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