Insights into Regulation Which way does the carbon go? Ultimately, biochemistry seeks to learn how a cell behaves; how pathways of synthesis and degradation are controlled and what determines carbon flux. Obviously, a need for ATP must be met by the cell making ATP, and ATP excess likewise must deter ATP synthesis. A pathway in a living system operates in a delicate balance that favors one direction but is poised to reverse or shutdown when a circumstances favor a change. A search for how this is achieved leads us directly into the realm of allosteric enzymes and hormonal effects on cells. How these agents change pathway direction is a fascinating insight into the autonomous control of living systems.

How are pathways regulated? Fortunately, we can perhaps list 3 principal ways. First, because many enzymes function at subsaturation with regard to substrate, increasing the substrate concentration by whatever means will increase the velocity in a forward direction (click 1). Second, many enzymes that regulate pathway flow are allosteric. This brings T- state and R-state transitions into the discussion and allows one to focus on metabolites whose increase in concentration could alter enzyme activity through structural transitions (click 1). Third, is covalent modification, which for our purposes, mostly involves adding phosphate groups to an enzyme, an action that can make the enzyme more active or less active (click 1). Covalent modification by phosphorylation is generally under the control of hormones that activate c-AMP which in turn activates protein kinase enzymes (click 1). Click 1 to go on. A  B [A] dB/dt ATP ADP PO 4  velocity T-state R-state Allosteric effector Hormone cAMP Protein kinase

In the case of glycogen, allostery and covalent modification play dominant roles in the reglulation of enzyme activity. Allosteric effects are seen in the T and R forms of glycogen phosphorylase (click 1). This is not to be confused with the a and b forms, which are covalently modified by phosphate groups on the protein (click 1). Note, there are 4 different structural forms of the dimeric protein phosphorylase. Click 1 to see which forms are active. Both T-a and R-a are active as well as R-b. R-b, however, depends on 5’-AMP bound to the molecule, which depends on the concentration of 5- AMP (click 1). T-a and R-a stay active as long as phosphate is bound,. R-b loses activity when 5’-AMP dissociates and T-a and R-a lose activity when phosphate is cleaved. b TR P P a P P T R Phosphorylase A second point that should not escape your notice is the position of the phosphate group in the R-a (click 1). Phosphate is buried within the protein and therefore more difficult to remove by a phosphatase enzyme. In the T form the phosphate group is exposed to the exterior of the protein (click1). Click 1 to go on. 5-AMP Active Less active Buried Exposed 5-AMP

Phosphatase Phosphatases counter the action of kinases by removing phosphate groups. These enzymes are subject to covalent modification, which means they too are controlled by hormones. Phosphatase regulation, however, depends on the tissue. In muscle, the phosphatase is bound to G protein, which is the target of kinase enzymes (click 1). The G protein has two sites for binding phosphate. Filling site 1 activates the phosphatase (click 1). Site 1 is filled by a kinase that responds to insulin (click 1). Alternatively, a c- AMP dependent protein kinase fills both sites, either by acting directly on the inactive complex (click 1) or just the second site (click 1). P P P Active G-protein Insulin Inactive G-proteinInactive phosphatase Inactive phosphatase Both lead to an inactive phosphatase that dissociates from the complex (click 1) cAMP kinase

Pyruvate Glucose 6-PO 4 Net carbon flux in glycolysis is controlled mainly by PFK-1 (click 1). PFK-1 is sensitive to allosteric effectors, the main one being fructose 2,6-bisPO 4 (click 1). The reverse reaction is catalyzed by fructose 1,6 bisphosphatase-1 (FBP-1) (click 1). High levels of the F2,6BP turn on glycolysis by activating PFK-1 and inhibiting FBP-1 (click 1). The result is a net flow of carbon from G-6-P to pyruvate (click 1). If, however, F2,6BP is destroyed by its phosphatase (FBP-2), the flow towards pyruvate is halted in intensity (click 1). A forced increase in the carbon flux from pyruvate to G-6-P occurs during gluconeogenesis (click 1). Stimulation occurs by activating PEPCK by a c-AMP kinase (click 1). This shows the importance of hormonal control, specifically glucagon, in promoting gluconeogenesis (click 1). Click 1 to go on. PFK-1 FBP-1 PEPOAA PEPCK Malate Protein kinase Glucagon CH 2 OH O 3 POCH 2 OPO 3 = O Fructose 2,6- bisPhosphate c-AMP Glycolysis-Gluconeogenesis F2,6BP

What have your learned? 1. Name a hormone that inhibits glycogen breakdown. Describe the mechanism. Insulin. Insulin stimulates phosphatases, one of which converts phosphorylase a to phosphorylase b 2. Name an allosteric effector that stimulates gluconeogenesis. None. Stimulation by allostery occurs only in glycolysis. 3. Predict the consequences to the cell by activating PFK-2. Glycolysis would be stimulated and gluconeogenesis would be suppressed. PFK-2 is the kinase that converts F6P into F2,6BP. 4.What would be the expected effect of insulin on gluconeogenesis. Justify you answer. Gluconeogenesis would be suppressed. One reasons is that insulin activates phosphatases. PEPCK would be a target for inactivation.