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Exposure of rabbits to cold or fasting results in marked increases in the number and size of liver lysosomes, associated with increases in the total and “free” proteolytic activity. Changes in lysosomal morphology also indicate increased activity and fragility. These effects of cold and fasting are correlated with decreases in the levels of liver fructose bisphosphatase (EC 3.1.3.11) activity and changes in the molecular properties of the purified enzyme, including the loss of a small peptide containing the only tryptophan residue in the peptide chain.

Digestion of native fructose 1,6-bisphosphatase (Fru-P2ase) with subtilisin determines the removal of a tryptophan-containing peptide from the amino-terminal region of each of the four subunits. This molecular effect is accompanied by changes in the functional properties of the enzyme, namely a shift of the pH optimum from 7.5 to 9.2 and a desensitization to allosteric inhibition by AMP.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) requires a free divalent metal and a metal chelate for optimum activity in the neutral pH range. The latter requirement can be satisfied by histidine, which appears to function as the chelating metabolite in vivo . Under fasting conditions, the concentrations of histidine in rabbit liver vary in the range required for activation of fructose bisphosphatase, and the enzyme itself is modified so that it becomes more responsive to histidine.

Rabbit liver fructose 1,6-bisphosphatase is converted by subtilisin to a form with smaller subunits and modified catalytic and allosteric properties. Analysis of the changes in the catalytic properties of the enzyme during digestion with subtilisin indicates that these properties depend on the presence of strong functional interactions between all four subunits in the molecule. On the other hand, sensitivity to inhibition by AMP appears to depend only on intrachain interactions. Changes in subunit interaction relating to relaxation in protein conformation during digestion with subtilisin were also inferred from the changes in concentration dependency for the effects of urea on the fluorescent emission spectrum. Structural changes around the region containing the single tryptophan residue appear to be related to the changes in catalytic properties.

During prolonged starvation, fructose 1,6bisphosphatase (EC 3.1.3.11) activity in rabbit liver and kidney shows a transient decrease during the first 36 hr, before rising at 96 hr to levels severalfold higher than those found in the livers of fed animals. Proteolytic activity appears in the 105,000 × g supernatant fraction within several hours of starvation, and continues to increase during the entire 96-hr period. On refeeding, the activities return to nearly the control levels within 24 hr. The catalytic properties of fructose 1,6-bisphosphatase isolated from the livers of fasted rabbits are similar to those of the enzyme from fed animals, but its structure is modified, since it no longer contains the single tryptophan residue located near the NH 2 -terminus in the native enzyme. Thus this tryptophan residue is not required for the neutral pH optimum. The structural changes and the transient decrease in activity may be related to the observed increase in “free” proteolytic activity.

Two lysosomal proteases have been detected, each capable of catalyzing a different modification of the NH2-terminal region of rabbit liver fructose bisphosphatase. Protease I has optimum activity near pH 5.0, in contrast to Protease II, which is most active only at lower pH. The peptide formed by the action of Protease I is acid-insoluble, with a molecular weight of approximately 7,000, whereas Protease II releases a small acid-soluble peptide, containing the tryptophan residue that is located near the NH2-terminus. In fasted rabbits, Protease II appears to be selectively released from the lysosomes.

Fructose 1,6-bisphosphatase undergoes specific conformational changes in the presence of the substrate fructose 1,6-bisphosphate and of the allosteric modifier, AMP and also on activation by cystamine. These changes can be monitored by observing the changes in sensitivity to digestion by subtilisin. In the presence of AMP the enzyme is protected against the action of subtilisin. Some protection is also observed with high concentrations of fructose bisphosphate while low concentrations of this substrate, which are ineffective alone, enhance the protective effect of low concentrations of AMP. The results suggest that AMP induces a resistant conformation, and that fructose bisphosphate promotes the binding of AMP. Divalent cations, although essential for activity, do not protect the enzyme against digestion by subtilisin. The native enzyme is activated by disulfide exchange with cystamine, and the activated enzyme is also more resistant to subtilisin. Thus, the enzyme in both inhibited (AMP) and activated conformations (cystamine) is rendered resistant to modification by proteolysis.

Removal of the NH 2 -terminal region of fructose 1,6-bisphosphatase from rabbit liver by digestion with subtillisin, or changes in conformation in this region of the protein produced by exposure to low concentrations of urea, result in similar changes in catalytic and allosteric properties of the enzyme. These changes include shift of the pH optimum to more alkaline pH, and loss of sensitivity to inhibition by AMP. The conformation changes are monitored by changes in the fluorescence of the single tryptophan residue, which is located near the NH 2 -terminus. Thus, the tryptophan-containing peptide appears to determine the functional properties of the native enzyme.

Seasonal variations in the properties of rabbit-liver fructose 1,6-bisphosphatase have now been linked to corresponding changes in the levels of proteolytic activity in the liver extracts. Incubation of native fructose 1,6-bisphosphatase with purified liver lysosomes causes a 3-fold increase in catalytic activity at pH 9.2, with a smaller, and variable, decrease in activity tested at pH 7.5. These changes in catalytic properties are accompanied by the appearance of a smaller subunit, as was previously reported for the enzyme treated with subtilisin. AMP, a negative modulator of fructose bisphosphatase activity, protects against this action of lysosomes. This proteolytic modification of fructose bisphosphatase by lysosomal enzymes may play a role in the modulation of gluconeogenesis.