Multi-enzyme complexes in thermophilic organisms: isolation and characterization of the pyruvate dehydrogenase complex from Thermus aquaticus AT62

Abstract

The pyruvate dehydrogenase complex from the thermophilic bacterium Thermus aquuticus was purified by Triton X-100 extraction and chromatography on phenyl-Sepharose CL-4B and HPLC-hydroxyapatite. The electrophoretic pattern of the purified enzyme complex was similar to that of the enzyme complex from Bacillus subtifis, with four bands: the a-chain (Mr 39600) and @-chain (Mr 37500) of the pyruvate dehydrogenase component, the dihydrolipoamide acetyltransferase component ( Mr 58500) and the dihydrolipoamide dehydrogenase component (Mr 53900). Antibodies against the purified T. aquuticus pyruvate dehydrogenase complex cross-reacted with the enzyme complex from B. subtilis and, to a minor extentiwith that from bovine heart. No cross-reactivity could be observed with the enzyme complex from Escherkhiu coli. The T. uquuticus enzyme complex had a temperature maximum at 72 OC. 2-Oxobutyrate was a poor substrate and other 2-oxoacids were competitive inhibitors of the overall reaction. Long-chain 2-oxoacids showed a greater inhibitory effect, possibly caused by hydrophobic interactions. GTP inhibited the enzyme activity. Regulation of the pyruvate dehydrogenase complex from T. aquaticus by allosteric mechanisms or by reversible phosphorylation could not be demonstrated.

Growth Rate-Dependent Modulation of Carbon Flux through Central Metabolism and the Kinetic Consequences for Glucose-Limited Chemostat Cultures of Corynebacterium glutamicum

“…Furthermore, this maximal rate of enzyme synthesis could not be increased by imposing a thiamine limitation, as has been observed for other microorganisms (6), although increased PDH concentrations were observed under such conditions at lower growth rates. In view of the lack of allosteric effectors (other than NADH 2 ) for the PDH of grampositive bacteria (16,31), the catabolic flux will be controlled by the enzyme concentration and the pyruvate pool concentration. The enzyme concentration profile is such that the pyruvate pool would logically increase as a function of the growth rate, possibly inducing the shift in anaplerotic reactions.…”