Posttranslational modification of 6-phosphofructo-1-kinase in Aspergillus niger

Abstract

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the Vmax of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.

FIG. 1.

Growth curve of A. niger mycelium in a minimal medium with ammonium ions as the sole nitrogen source. Error bars indicate standard errors of the means.

FIG. 2.

SDS-PAGE of partially purified PFK1 from the mycelium at different ages, after elution from the affinity column. In the left lane of each gel the following standards are shown: bovine serum albumin (molecular weight, 66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), and trypsin inhibitor (20,000) (all purchased from Sigma).

FIG. 3.

SDS-PAGE of the partially purified native PFK1 protein digested with specific proteases. Subtilisin A (left panel) and proteinase A (middle panel) were used at 250 μg/ml, while proteinase K (right panel) was used at a lower concentration (25 μg/ml). The same standard markers as in Fig. 2 were used.

FIG. 4.

When the native PFK1 was subjected to treatment with proteinase K (25 μg/ml) and the activity was measured during the incubation, virtually all activity was lost within 1 h. After addition of the serine protease inhibitor PMSF (1 mM) and the catalytic subunit of cAMP-dependent protein kinase (PKA) (10 U) to the system (arrow), the activity gradually reestablished. The activities were measured first with 0.1 mM ATP, then ATP was added to a final concentration of 1 mM, and finally fructose-2,6-biphosphate (F-2,6-P) was introduced into the system to 8 μM. The initial substrate (fructose-6-phosphate) concentration was 8 mM. The results are the means ± standard errors of the means from triplicate determination.

FIG. 5.

Hill plot of PFK1 activities of the fragmented enzyme (•) and native protein (▪) determined at different fructose-6-phosphate (F6P) concentrations without any effectors present in the measuring system.

FIG. 6.

Kinetic parameters of the native PFK1. (A) ATP profile of the native enzyme as detected in the presence of 8 mM fructose-6-phosphate in the system with no other effectors added. (B) Activities measured at different concentrations of fructose-2,6-biphosphate (F26P).

FIG. 7.

Kinetic parameters of the partially purified 49-kDa fragment. (A) ATP profile of the fragment as detected in the presence of 8 mM fructose-6-phosphate in the system with and without 4 μM fructose-2,6-biphosphate (F-2,6-P) present. (B) Activities measured at different concentrations of fructose-2,6-biphosphate (F26P).