Theoretical Study of the Catalytic Mechanism of E1 Subunit of Pyruvate Dehydrogenase Multienzyme Complex from Bacillus stearothermophilus

Biochemistry ◽  
2013 ◽  
Vol 52 (45) ◽  
pp. 8079-8093 ◽  
Author(s):  
Xiang Sheng ◽  
Yongjun Liu
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Catalytic Mechanism ◽  
Theoretical Study ◽  
Multienzyme Complex

scholarly journals Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly

1995 ◽  
Vol 306 (3) ◽  
pp. 727-733 ◽  
Author(s):  
I A D Lessard ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Self Assembly ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Decarboxylase ◽  
Binding Domain ◽  
Alpha Subunit ◽  
Multienzyme Complex ◽  
Beta 2 ◽  
Dihydrolipoyl Acetyltransferase

The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.

scholarly journals Lipoylation of the E2 components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli

1991 ◽  
Vol 277 (1) ◽  
pp. 153-158 ◽  
Author(s):  
L C Packman ◽  
B Green ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Catalytic Mechanism ◽  
Chemical Reduction ◽  
Pyruvate Dehydrogenase Complex ◽  
Exchange Chromatography ◽  
Lysine Residue ◽  
Multienzyme Complex ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The number of functional lipoyl groups in the dihydrolipoyl acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been re-assessed by means of a combination of protein-chemical and mass-spectrometric techniques. (1) After the complex had been treated with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, the lipoyl domains were excised from the complex, treated with NaBH4 and re-exposed to N-ethyl[2,3-14C]maleimide. All the chemically reactive lipoyl groups in the native complex were found to be catalytically active. (2) Proteolytic digests of the separated lipoyl domains were examined for the presence of the lipoylation-site peptide, GDKASME, with and without the lipoyl group in N6-linkage to the lysine residue. Only the lipoylated form of the peptide was detected, suggesting that all three lipoyl domains are fully substituted at this site. (3) The behaviour of each lipoyl domain was examined on ion-exchange chromatography in response to alkylation with 4-vinylpyridine after either chemical reduction of the lipoyl group with dithiothreitol or reductive acetylation by the pyruvate dehydrogenase complex in the presence of pyruvate. All three domains exhibited a quantitative shift in retention time, confirming that each domain was fully substituted by an enzymically reactive lipoyl group. (4) When subjected to electrospray mass spectrometry, each domain gave a mass consistent with a fully lipoylated domain, and no aberrant substitution of the target lysine residue was detected. The same result was obtained for the lipoyl domain from the E. coli 2-oxoglutarate dehydrogenase complex. (5) Previous widespread attempts to assess the number of functional lipoyl groups in the pyruvate dehydrogenase multienzyme complex, which have led to the view that a maximum of two lipoyl groups per E2 chain may be involved in the catalytic mechanism, are in error.

Sign in / Sign up

Export Citation Format

Share Document