[4] ADP-dependent glucokinase and phosphofructokinase from Pyrococcus furiosus

Publisher Summary ADP-dependent glucokinase and phosphofructokinase were demonstrated for the first time by Kengen et al . in the hyperthermophile Pyrococcus furiosus . This anaerobic archaeon grows optimally at 100° by fermenting sugar polymers and polypeptides to mainly acetate and alanine. The catabolism of sugars, such as starch, laminarin, maltose, or cellobiose, has been investigated in detail and has led to the discovery of novel type Embden–Meyerhof pathway, involving several enzymatic steps that are different from the classical ones. In addition to ADP-dependent kinases, the pathway involves a one-step, ferredoxin-dependent conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate instead of the conventional two-step, NAD-dependent and ATP-generating conversion. Furthermore, a remarkable AMP-dependent, ATP-generating pyruvate kinase has been described, which is different from the normal ADP-dependent pyruvate kinase. Moreover, the available P. furiosus genome sequence shows that for several other glycolytic enzymes, no homologous sequences can be identified, suggesting that these enzymes may be different from the classical ones as well. This chapter describes the procedures for assay and purification of both kinases and the cloning and expression of their genes.