CARBOHYDRATE-METABOLISM IN THERMOPROTEUS-TENAX - IN-VIVO UTILIZATION OF THE NON-PHOSPHORYLATIVE ENTNER-DOUDOROFF-PATHWAY AND CHARACTERIZATION OF ITS FIRST ENZYME, GLUCOSE-DEHYDROGENASE

Thermoproteus tenax is a hyperthermophilic, falcultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathw ays, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the mod ified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investig ated and the first enzyme of the ED pathway, glucose dehydrogenase, wa s characterized. The distribution of the C-13 label in alanine synthes ized by cells grown with [1-C-13]glucose indicated that in vivo the EM P pathway and the modified ED pathway operate parallel, with glucose m etabolization via the EMP pathway being prominent, To initiate studies on the regulatory mechanisms governing carbon flux via these pathways , the first enzyme of the ED pathway, glucose dehydrogenase, was purif ied to homogeneity and its phenotypic properties were characterized, T he pyridine-nucleotide-dependent enzyme used both NAD(+) and NADP(+) a s cosubstrates, showing a 100-fold higher affinity for NADP(+). Beside s glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the en zyme is the oxidation of glucose by NADP(+), A striking feature was th e influence of NADP(+) and NAD(+) on the quaternary structure and acti vity state of the enzyme. Without cosubstrate, the enzyme was highly a ggregated (mol. mass > 600 KDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically activ e, homomeric dimers with a mel, mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarit y to the respective partial sequences of alcohol dehydrogenases and so rbitol dehydrogenases, but no resemblance to the known pyridine-nucleo tide-dependent archaeal and bacterial glucose dehydrogenases.