The mechanism by which glucose inhibits β-galactosidase synthesis remained a complete mystery for about 20 years after Monod first observed the phenomenon. Richard S. Makman and Earl W. Sutherland found an important clue to the mystery in 1965 when they observed that the intracellular concentration of 3', 5'-cyclic adenylate or cAMP drops from about 10-4 M to 10-7 M when glucose is added to a growing culture of E.coli.
Genetic studies confirmed cAMP's involvement in catabolite repression. Two mutant classes were isolated that could not synthesize lac enzymes when cultured in a medium containing lactose but no glucose. Class mutants regained the ability to synthesize lac enzymes when cAMP was added to the growth medium but class ‖ mutants did not.
Subsequent studies showed that class I mutants have defects in adenylate cyclase, the enzyme that converts ATP to cAMP. The structural gene for adenylate cyclase, cya, maps at minute 85.98. Adenylate cyclase exists in an active form that is phospfborylated, (it contains an attached phosphate group) and an inactive form that is dephosphorylated. Class ‖ mutants have defects in a protein that binds cAMP. This protein, called the cAMP receptor protein (CRP) or the catabolite activator protein (CAP), is encoded by the crp gene, which maps at minute 75. 09. In vitro studies have shown that CRP and cAMP form a complex, denoted cAMP·CRP complex, which is needed to activate the lac system. The cAMP · CRP requirement is independent of the repression system since cya and crp mutants cannot make lac mRNA even if a lacI- or lacOc mutation is present. Thus, cAMP · CRP is a positive regulator or activator, in contrast to the repressor and the lac operon is independently regulated both positively and negatively.
Based on the information presented above, it seems reasonable to propose that glucose somehow inhibits phosphorylation of adenylate cyclase, thereby preventing cAMP formation. The next challenge was to find the link between glucose metabolism and adenylate kinase phosphorylation. Biochemical and genetic studies indicated that the link is a glucose-specific, phosphoenolpyruvate-dependent phosphotransferase system (PTS). This system uses energy supplied by phosphoenolpyruvate (PEP) to phosphorylate glucose as it transports the sugar across the inner cell membrane.
The system requires four proteins. Two of these, enzyme I (E1) and the histidine-containing protein (HPr), are also components of other sugar transport systems and therefore are unlikely to be direct participants in a glucose-specific phenomenon. The two other proteins, enzyme IIA (EIIA) and enzyme IIBC (EIIBC) are specific for the glucose transport system and therefore more likely participants in a glucose-specific phenomenon.
EIIA participates in catabolite repression by two different mechanisms. The first mechanism is based on the fact that EIIA can transfer a phosphoryl group from HPr-P to either EIIBC or adenylate cyclase. However, the preferred substrate is EIIBC, which then transfers the phosphoryl group to glucose to form glucose-6-phosphate. When glucose is unavailable EIIBC will be fully phosphorylated and EIIA-P has no other alternative but to transfer its phosphoryl group to adenylate cyclase. Phosphorylation changes the inactive dephosphorylated form of adenylate cyclase to the active phosphorylated form of adenylate cyclase, which then converts ATP to cAMP. Thus, glucose interferes with the conversion of the inactive form of adenylate cyclase to the active form.