Lecture 14: Regulation of Proteins 1: Allosteric Control of ATCase Overview of Regulatory Mechanisms Description of ATCase Allosteric Properties of ATCase

Biological Processes are Carefully Regulated Allosteric Control: The activity of some proteins can be controlled by modulating the levels of small signalling molecules. The binding of these molecules causes conformational changes in the protein which affect its activity. Multiple forms of Enzymes: Different tissues or developmental stages sometimes have specific versions of a given enzyme which have distinct properties although they may have the same basic activity. Reversible Covalent Modification: The activity of many proteins is controlled by attachment of small chemical groups. The most common such modification is phosphorylation- attachment of a phosphate group. Proteolytic Activation: Some enzymes are synthesized in an inactive form and must be activated by cleavage of the inactive form.

Allosteric Regulation Allosteric enzymes have multiple subunits which exert influence on one another in the complex. The binding of substrate at one site affects the affinity for substrates at other sites, eventually causing a conformational shift from a less activate state to a more active state. (cooperativity) Allosteric enzymes don’t follow Michaelis-Menten kinetics. The activity increases steeply above a “threshold” so that a small change in [S] causes a large change in activity. Small molecule regulators can bind to the enzyme and change the threshold, so as to adjust the activity to the required level. With Inhibitor With Activator Less Active State More Active State

Aspartate Transcarbamoylase Aspartate transcarbamoylase, or ATCase, catalyses the first step in a biosynthetic pathway that produces pyrimidine nucleotides (eg CTP) needed for nucleic acids, energy storage, and enzyme cofactors. ATCase Many more enzymes…

CTP ATCase is inhibited by the end-product of its pathway ATCase Aspartate & Carbamoyl phosphate CTP (doesn’t resemble substrates of ATCase) This is an example of feedback inhibition. When CTP is abundant, the pathway is shut down, but when CTP levels are low and more is needed, the activity of ATCase increases to make more CTP.

c r Quaternary Structure of ATCase ATCase has two subunit types: c subunit (catalytic subunit; 34 kD) which forms trimers r subunit (regulatory subunit; 17 kD) which forms dimers These can be dissociated, isolated, and reconstituted. Active Complex: c6r6 c Catalytic Subunit: r Regulatory Subunit:

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Identification of Active Sites using an Inhibitor The compound PALA is a structural mimic of an intermediate in the reaction. X-ray crystallography reveals that it binds at the active site in between 2 catalytic subunits.

PALA Binding causes a Conformational Change Two distinct quaternary forms exist in equilibrium. PALA stabilizes the more active R state. T state for “tense” predominates in absence of substrate. R state for “relaxed” predominates in presence of substrate.

CTP Binding inhibits the Conformational Change CTP binds in regulatory sites on the r subunits, distant from the active sites. The allosteric inhibitor CTP shifts the equilibrium toward the less active T state.

The R to T transition is All-or-Nothing In ATCase, a given complex is either in the R state or T state- there are no “mixed” complexes. But there is a mixed population of T-state complexes and R-state complexes at equilibrium.

[T]eq R T = 200 = Keq [R]eq RT ln ( Keq ) Thermodynamics of the Allosteric Transition In the absence of substrate or regulators, the T state is about 200 times as prevalent as the R state. (at equilibrium) [T]eq R T = 200 = Keq [R]eq R state RT ln ( Keq ) T state = 13.1 kJ/mol So only a small energy difference exists between the T and R states. The binding of effectors could easily modulate this energy difference.

ATCase does not follow Michaelis-Menten Kinetics ATCase shows a sigmoidal rate profile.

T R ATCase switches between T and R states At low substrate concentrations, the enzyme is primarily in the less active T state. But as [substrate] increases, more of the complexes switch to the more active R state. T R (T state curve is identical to isolated catalytic subunits) High Km Low Km

T R CTP acts as an Allosteric Inhibitor CTP acts to shift the equilibrium towards the T states, favoring the High Km form of the enzyme and reducing the overall activity. CTP T R High Km Low Km

T R ATP acts as an Allosteric Activator CTP acts to shift the equilibrium towards the R states, favoring the low Km form of the enzyme and increasing the overall activity. ATP T R High Km Low Km

Binding of Effectors Adjusts the Equilibrium between T and R States [T]eq [R]eq R T In the presence of ATP a higher percentage of complexes are in the T state. In the presence of CTP a lower percentage of the complexes are in the R state. = [S] / (Km of R state)

The binding of the effector influences the binding of substrate ATP Allosteric Equilibrium R T CTP R + S RS Substrate- Binding Equilibrium CTP ATP T + S TS

Physiological Role of CTP and ATP Regulation of ATCase CTP is a feedback inhibitor. When CTP levels are high, it is unnecessary to make more pyrimidines, so the inhibition of ATCase slows down the pathway. When CTP levels fall, the inhibition is removed, and more pyrimidines can be synthesized. ATP is a purine nucleotide and is not a product of the ATCase pathway. ATP is the major cellular energy source and if ATP levels are high, the cell is metabolically very active and preparing to divide. Therefore it must duplicate its DNA, and both ATP and CTP are needed for DNA synthesis. So high ATP levels can override the inhibitory affects of CTP.

Summary: Aspartate transcarbamoylase (ATCase) is an allosteric enzyme which carries out the first step in the synthesis of pyrimidine nucleotides. Allosteric enzymes use changes in conformation to switch between different states which have different levels of activity. Binding of allosteric effectors can control the switch between states, and thereby increase or decrease the enzyme activity to exert control over biological processes. Key Concepts: Types of Regulation Feedback inhibition Allosteric transition in ATCase ATP and CTP as allosteric effectors of ATCase