초록
<P>Cofactor specificities of glycolytic enzymes in <I>Clostridium thermocellum</I> were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PP<SUB>i</SUB>)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the <I>C. thermocellum</I> genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (<I>ppdk</I>) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the <I>ppdk</I> gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of <I>C. thermocellum</I>. The requirement for PP<SUB>i</SUB> in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PP<SUB>i</SUB>-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PP<SUB>i</SUB> must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PP<SUB>i</SUB> generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol.</P>