PHOSPHOGLYCERATE KINASE AND TRIOSEPHOSPHATE ISOMERASE FROM THE HYPERTHERMOPHILIC BACTERIUM THERMOTOGA-MARITIMA FORM A COVALENT BIFUNCTIONALENZYME COMPLEX

Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium The rmotoga maritima has been purified to homogeneity. A second larger enz yme with PGK activity and identical N-terminal sequence was also found . Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity. No other TIM is detectable in T.maritima crude extracts. As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas t he bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kD a. SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein. Both enzymes show high thermostability. Measurements of the catalytic properties revealed no extraordinary results. pH optima, K-m values a nd activation energies were found to be in the range observed for othe r PGKs and TIMs investigated so far. The corresponding pgk and tpi gen es are part of the apparent gap operon of T.maritima. This gene segmen t contains two overlapping reading frames, where the 43 kDa PGK is enc oded by the upstream open reading frame, the pgk gene. On the other ha nd, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk ge ne and the overlapping downstream open reading frame of the tpi gene. A programmed frameshift may be responsible for this fusion. A comparis on of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similar ity to the corresponding enzymes from different procaryotic and eucary otic organisms.