Chem*3560 Lecture 6: Allosteric regulation of enzymes Metabolic pathways do not run on a continuous basis, but are regulated according to need Catabolic pathways run if there is demand for ATP; for example glycolysis may be slowed down if beta oxidation is meeting current energy needs. Beta oxidation is more suited for slow steady delivery of energy. Glycolysis supports need for rapid delivery of energy. Pathways for biosynthesis are also controlled Is there a need for the product? Is there sufficient energy or substrate or reducing power to carry out the synthesis? Where there are parallel catabolic and synthetic pathways, regulation is especially important, because if both are allowed to run concurrently, it wastes energy: Glycolysis: gain 2 ATP and 2 NADH Glucose 2 pyruvate Gluconeogenesis: cost 6 ATP + 2 NADH If equivalent amounts of substrate are allowed to pass in each direction, 4 ATP would be hydrolysed with no net benefit. Glycolysis and gluconeogenesis are regulated in a reciprocal fashion so that each pathway is only active if required, and then the opposite pathway is shut down. Pathways are typically regulated near their starting point Regulation near the start prevents substrate from being committed to the pathway when product is not needed. If the pathway branches to yield two alternative products, then the branch point is regulated so that only the product that is need is produced. Typically an end product serves as the regulating ligand, a process known as negative feedback. 1 2 3 4 A! B! C!D E Reaction 1 is inhibited by product E
Enzymes may be regulated in several ways: Allosteric regulation of substrate affinity; Turn on or off by covalent modification of the enzyme; Turn on by regulated expression of gene (transcription) or of synthesis of enzyme (translation); Turn off by regulated destruction of the enzyme (the amino acids are recycled and used for other purposes). The first two effects tend to give immediate responses to changing conditions. The last two methods provide longer term responses. The enzyme aspartate transcarbamoylase (ATCase) is a classic case of allosteric regulation of affinity ATCase catalyzes an early step in synthesis of pyrimidines ATCase more steps carbamoyl phosphate + aspartate carbamoyl aspartate uridine, cytidine ATCase is negatively regulated by CTP If CTP is present, there's no need to make more This is an example of negative feedback control. ATCase is positively regulated by ATP If ATP is abundant, cells are growing, DNA and RNA synthesis are active If ATP is abundant and [ATP] > [CTP], it's time to make more pyrimidines to match the availability of purines.
ATCase shows sigmoidal kinetics with respect to substrate aspartate Allosteric kinetics is described in terms of a low affinity T-state and high affinity R-state When [Asp] is low, the enzyme is in T-state; as [Asp] increases, the enzyme switches to a high affinity R-state. The enzyme is monitored in terms of the fraction of max rate of catalysis, v o /V max, which is essentially a measure of the occupancy of the catalytic site. With substrates only, the reaction rate follows sigmoidal curve C. When CTP binds (curve D), this favours the T-state, so the curve is shifted to the right (higher [Asp] is needed to get the enzyme going). When ATP binds (curve B), this favours the R-state, so the curve is shifted to the left (less [Asp] needed). If [ATP] is high enough (curve A), there is no T-state, and ATCase follows the hyperbolic curve of the pure R-state. For allosteric enzymes, the substrate concentration giving v o /V max = 0.5 is designated as K' or K 0.5, and represents the apparent substrate affinity, just as P 50 represents O 2 affinity for hemoglobin. Although K M is derived in a similar manner, the term K M is not used for allosteric enzymes because they don't follow the Michaelis-Menten equation. T R switch induced by substrate occupancy is called the positive homotropic effect (the basic sigmoidal curve C above). Homotropic means literally "change induced by the same substance", positive because affinity increases with increasing [S],
ATP and CTP are allosteric effectors for ATCase T R switch induced by binding ATP is called a positive heterotropic effect, and shifts the curve left.. Heterotropic means literally "change induced by the another substance" (other than a substrate). R T switch induced by binding CTP is called a negative heterotropic effect; and shifts the curve right. Negative because affinity decreases with increasing [CTP]. ATP and CTP are allosteric effectors, substances that do not participate directly in the catalytic reaction, but which modulate the allosteric behaviour of the enzyme to regulate the reaction. When ATP and CTP are both present, they compete for the same binding site (see below), and the relative concentration of the two nucleotides will determine whether a positive or negative effect is observed. CTP has slightly more affinity, so [ATP] has to be significantly higher than [CTP] for a positive effect. Allosteric effectors are much more versatile than simple competitive or noncompetitive inhibitors. When substrate concentration [S] is close to the enzyme's K 0.5, a positive effector can induce nearly full activity, while a negative effector can virtually turn the enzyme off, exactly as desired for metabolic regulation. ATCase is composed of two kinds of subunits c (catalytic) subunits (33 kda each) contain the substrate binding and catalytic site of the enzyme, and can be in T or R catalytic states. r (regulatory) subunits (17 kda each) contain the ATP/CTP binding site and control the activity of c subunits by determining their tendency to T or R state. r subunits have no inherent catalytic activity.
The complete ATCase molecule has formula c 6r 6. This can be broken down into two modules of c 3 (three catalytic sites) and three modules of r 2. Each trimeric catalytic module contains c subunits placed every 120 o around a central axis (only one is shown in the figure). On its own, the c 3 module is catalytically active, but unregulated, and with normal Michaelis-Menten hyperbolic kinetics. The r 2 module binds ATP or CTP, but has no catalytic activity of its own. The r 2 modules bridge between the c subunits of the upper catalytic trimer and the lower catalytic trimer. The structure above is the R state with substrates and ATP bound. Below it is the T state with sites vacant. In the T-state, the r 2 modules reorient themselves and bring the catalytic units closer together. This causes interference between projecting loops of the catalytic subunits.