Biochemical Properties and the Physiological Role of the Fructose-1,6-Bisphosphate Activated l-Lactate Dehydrogenase from Staphylococcus epidermidis

Abstract

Staphylococcus epidermidis ATCC 14990 possesses an l-lactate dehydrogenase dependent on nicotinamide adenine dinucleotide (NAD) which is activated by fructose 1,6-bisphosphate (fructose-1,6-P2). Molecular weight studies with purified l-lactate dehydrogenase showed that fructose-1,6-P2 is necessary to stabilise the tetrameric form, having a molecular weight of about 130000. Dialysation of the enzyme led to a dissociation into subunits which could reasociate with fructose-1,6-P2 to the tetramer. The dialysed enzyme also exhibited a lag-period in the progress curve obtained by plotting the dependence of velocity versus time. This lag-period is absent when the enzyme was preincubated with fructose-1,6-P2. The lactate dehydrogenase exhibited only pyruvate reduction activity; the reverse reaction (l-lactate oxidation) could not be demonstrated in the enzyme assay system. Physiological studies demonstrated that the intracellular concentration of fructose-1,6-P2 influences the lactate dehydrogenase activity. The influence of the intracellular level of fructose-1,6-P2 on the active state of the lactate dehydrogenase could be shown in several ways. A high intracellular fructose-1,6-P2 level, present in cells grown in a glucose-excess medium under anaerobic growth conditions, is correlated with an active state of the lactate dehydrogenase, which means that the lactate dehydrogenase in the cell-free extract was saturated with fructose-1,6-P2. The addition of fructose-1,6-P2 to the assay system could not increase the lactate dehydrogenase activity. In cells grown in a glucose-limited medium under anaerobic conditions, the fructose-1,6-P2 pool was exhausted due to glucose limitation. In this case, the lactate dehydrogenase was not fully saturated with fructose-1,6-P2 because the lactate dehydrogenase activity from the cell-free extract could be increased about 10-fold by addition of fructose-1,6-P2 to the assay system. A similar situation is present with cells grown under aerobic conditions in a glucose-excess medium. Here too, no intracellular fructose-1,6-P2 was detected and the active state of lactate dehydrogenase was markedly reduced. Glucose degradation studies with resting cell cultures under both anaerobic and aerobic conditions showed that only under anaerobic conditions a high intracellular level of fructose-1,6-P2 was present and a high amount of l-lactate was produced.