α-Keto acid dehydrogenase complexes. XIV. Regulation of the activity of the pyruvate dehydrogenase complex of Escherichia coli

Biochemistry ◽  
1970 ◽  
Vol 9 (6) ◽  
pp. 1434-1439 ◽  
Author(s):  
Edith Richmond Schwartz ◽  
Lester James Reed
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Keto Acid ◽  
Acid Dehydrogenase ◽  
Dehydrogenase Complex

scholarly journals Selective inactivation of the transacylase components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli

1976 ◽  
Vol 155 (2) ◽  
pp. 419-427 ◽  
Author(s):  
J P. Brown ◽  
R N. Perham
Keyword(s):  
Escherichia Coli ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Simple Method ◽  
Nitrobenzoic Acid ◽  
Acid Dehydrogenase ◽  
Lipoamide Dehydrogenase ◽  
Dehydrogenase Complex ◽  
Covalently Bound

1. The reaction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with maleimides was examined. In the absence of substrates, the complex showed little or no reaction with N-ethylmaleimide. However, in the presence of pyruvate and N-ethylmaleimide, inhibition of the pyruvate dehydrogenase complex was rapid. Modification of the enzyme was restricted to the transacetylase component and the inactivation was proportional to the extent of modification. The lipoamide dehydrogenase activity of the complex was unaffected by the treatment. The simplest explanation is that the lipoyl groups on the transacetylase are reductively acetylated by following the initial stages of the normal catalytic cycle, but are thereby made susceptible to modification. Attempts to characterize the reaction product strongly support this conclusion. 2. Similarly, in the presence of N-ethylmaleimide and NADH, much of the pyruvate dehydrogenase activity was lost within seconds, whereas the lipoamide dehydrogenase activity of the complex disappeared more slowly: the initial site of the reaction with the complex was found to be in the lipoyl transacetylase component. The simplest interpretation of these experiments is that NADH reduces the covalently bound lipoyl groups on the transacetylase by means of the associated lipoamide dehydrogenase component, thereby rendering them susceptible to modification. However, the dependence of the rate and extent of inactivation on NADH concentration was complex and it proved impossible to inhibit the pyruvate dehydrogenase activity completely without unacceptable modification of the other component enzymes. 3. The catalytic reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) by NADH in the presence of the pyruvate dehydrogenase complex was demonstrated. A new mechanism for this reaction is proposed in which NADH causes reduction of the enzyme-bound lipoic acid by means of the associated lipoamide dehydrogenase component and the dihydrolipoamide is then oxidized back to the disulphide form by reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). 4. A maleimide with a relatively bulky N-substituent, N-(4-diemthylamino-3,5-dinitrophenyl)maleimide, was an effective replacement for N-ethylmaleimide in these reactions with the pyruvate dehydrogenase complex. 5. The 2-oxoglutarate dehydrogenase complex of E. coli behaved very similarly to the pyruvate dehydrogenase complex, in accord with the generally accepted mechanisms of the two enzymes. 6. The treatment of the 2-oxo acid dehydrogenase complexes with maleimides in the presence of the appropriate 2-oxo acid substrate provides a simple method for selectively inhibiting the transacylase components and for introducing reporter groups on to the lipoyl groups covalently bound to those components.

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