Chapter 9 - Enzymes: Regulation of Activities

Factors underlying the widespread use of protein phosphorylation-dephosphorylation (2)

*(1): Mechanism for regulatory control*: the ease of interconversion of enzymes between their phospho- and dephospho- forms *(2) Chemical properties of the phosphoryl group itself*: the high charge density of protein-bound phosphoryl groups, generally -2 at physiologic pH, their propensity to form strong salt bridges with *arginyl and lysyl residues*, and their high exceptional *hydrogen-bonding capacity* renders them potent agents for modifying protein structure and function. Phosphorylation generally influences an enzyme's intrinsic catalytic efficiency or other properties by inducing conformational changes. Consequently, *the aa modified by phosphorylation can be and typically are relatively distant from the catalytic site itself*

Name the two most common covalent modifications that regulate protein function

- Phosphorylation-dephosphorylation - Acetylation- deacetylation.

Proteolytic subunits

A single proteasome molecule that assembles (or "coassembles") with other proteasome molecules to form a proteasome complex - The active sites of its proteolytic subunits face the interior of the cylinder, thus preventing indiscriminate degradation of cellular proteins.

Protein acetylation

A ubiquitous modification of metabolic enzymes - Thousands of other mammalian proteins are subject to modification by covalent acetylation, including nearly every enzyme in such core metabolic pathways as *glycolysis, glycogen synthesis, gluconeogenesis, the tricarboxylic acid cycle, β-oxidation of fatty acids, and the urea cycle* - Include many metabolically important enzymes, such as *acetyl-CoA synthetase, long-chain acyl-CoA dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, glutamate dehydrogenase, carbamoyl phosphate synthetase, and ornithine transcarbamoylase* - *Lysine acetyltransferases* catalyze the transfer of the acetyl group of acetyl-CoA to the ε-amino groups of lysyl residues, forming N-acetyl lysine (including histones) - Increases the steric bulk of the lysine side chain and transforms a basic and potentially positively charged primary amine into a neutral, nonionizable amide - Some proteins, particularly those in the mitochondria, become acetylated by reacting with acetyl-CoA directly, that is, without the intervention of an enzyme catalyst - Histone deacetylases and sirtuins, two classes of protein deacetylases

Why is metabolite flow said to be unidirectional

Bc while all chemical reactions are to some extent reversible, in living cells the *reaction products of one enzyme- catalyzed reaction serve as substrates for, and are removed by, other enzyme-catalyzed reactions* - This succession of coupled enzyme-catalyzed reactions is accompanied by an overall change in free energy that favors unidirectional metabolite flow - Water through a pipe (hydrostatic) analogy ; flow of water through the pipe remains unidirectional despite the presence of bends or kinks, which simulate steps with a small or even an unfavorable change in free energy, due to the overall change in height (KE), which corresponds to the pathway's overall change in free energy

Regulation of metabolite flow

Can be active or passive. - Passive: responses to changes in substrate level for coordination metabolite flow - Active: the mechanisms that regulate enzyme efficiency in response to internal and external signals

Regulatory covalent modifications

Can be reversible or irreversible - Eg. partial proteolysis and phosphorylation; frequently employed to regulate the catalytic activity of enzymes - Eg. acetylation, methylation, ADP- ribosylation, as well as phosphorylation; histones and other DNA-binding proteins in chromatin

What reduces/increases the rate metabolite flux

Catalytic efficiency or the quantity of the catalyst participating in the "bottleneck" or rate-limiting reaction will immediately reduce/increase metabolite flux through the entire pathway - As natural "governors" of metabolic flux, the enzymes that catalyze rate-limiting steps also constitute promising drug targets (eg. statin drugs)

"Ubiquination" catalysis

Catalyzed by a large family of enzymes called *E3 ligases*, which attach ubiquitin to the side-chain amino group of lysyl residues on their targets.

Intracellular E3 ligases

Catalyzes "ubiquination". Attaches ubiquitin to the side-chain amino group of lysyl residues on their targets. - Have the ability to discriminate between the different physical or conformational states of target proteins

Control of enzyme synthesis

Depends on the presence of: *(1) inducers*: typically substrates or structurally related compounds that stimulate the transcription of the gene that encodes them, or *transcription factors*, controlled by the hormones and other extracellular signals and their corresponding cell receptors. - Inducible enzymes of humans include tryptophan pyrrolase, threonine dehydratase, tyrosine-α-ketoglutarate aminotransferase, enzymes of the urea cycle, HMG-CoA reductase, Δ-aminolevulinate synthase, and cytochrome P450. *(2) Repression*: an excess of a metabolite which may curtail synthesis of its cognate enzyme Both induction and repression involve *cis elements*, specific DNA sequences located upstream of regulated genes, and *trans-acting regulatory proteins*

Reversible covalent modification of enzymes

Covalent modifications that regulate protein function. The most common ones by far are *phosphorylation-dephosphorylation* and *acetylation- deacetylation* - *Kinases*: phosphorylate proteins by *catalyzing transfer of the terminal phosphoryl group of ATP to the hydroxyl groups of seryl, threonyl, or tyrosyl residues*, forming O-phosphoseryl, O- phosphothreonyl, or O-phosphotyrosyl residues, respectively - The unmodified form of the protein can be regenerated by *hydrolytic removal of phosphoryl groups*, a thermodynamically favorable reaction catalyzed by *protein phosphatases*, aka: dephosphorylation - A typical mammalian cell possesses thousands of phosphorylated proteins and several hundred protein kinases and protein phosphatases that catalyze their interconversion - The ease of interconversion of enzymes between their phospho- and dephospho- forms accounts, in part, for the frequency with which both are utilized as a *mechanism for regulatory control* - Covalent phosphorylation persists only as long as the affected functional properties of the modified protein serve a specific need. Once the need has passed, the enzyme can be converted back to its original form - Potent agents for modifying protein structure and function due to their propensity to form strong salt bridges with *arginyl and lysyl residues*, and their *high exceptional hydrogen-bonding capacity* - Protein phosphorylation-dephosphorylation generally targets an early enzyme in a protracted metabolic pathway, as in feedback inhibition. However, phosphorylation-dephosphorylation involves one or more protein kinases and protein phosphatases, and is generally under direct neural and hormonal control - Acetylation-deacetylation targets multiple proteins in a pathway

Histone deacetylases

Deacetylase enzyme that catalyzes the removal by hydrolysis of acetyl groups, regenerating the unmodified form of the protein and acetate as products

Sirtuins

Deacetylase enzyme that uses NAD+ as substrate, which yields O-acetyl ADP-ribose and nicotinamide as products in addition to the unmodified protein

Promotes lysine acetylation.

Energy status of the cell, the high levels of acetyl-CoA (the substrate for lysine acetyltransferases and the reactant in nonenzymatic lysine acetylation) present in a well-nourished cell - When nutrients are lacking, acetyl-CoA levels drop and the ratio of NAD+/NADH rises, favoring protein deacetylation.

Compatmentalization

Ensures metabolic efficiency and simplifies regulation - Anabolic and catabolic pathways (in eukaryotes) that synthesize and break down common biomolecules often are physically separated from one another. - Eg. *fatty acid biosynthesis* occurs in the cytosol, whereas *fatty acid oxidation* takes place within mitochondria while many degradative enzymes are contained inside organelles called lysosomes - Eg. the ability of enzymes to discriminate between the structurally similar coenzymes *NAD+ and NADP+*, both with similar reduction potentials. However, most of the rxns that generate electrons destined for the ETC reduce NAD+, while enzymes that catalyze the reductive steps in many biosynthetic pathways generally use NADPH as the electron donor. - Eg. *glycolysis*, the breakdown of glucose to form two molecules of pyruvate, has a favorable overall ΔG ( -96 kJ/mol) too large to simply operate in "reverse" in order to convert excess pyruvate to glucose. Consequently, *gluconeogenesis* proceeds via a pathway in which the three most energetically disfavored steps in glycolysis are circumvented using alternative, thermodynamically favorable reactions catalyzed by distinct enzymes - Catalysts act bidirectionally, depending on the ratio of substrates to products. However, virtually all metabolic pathways possess one or more steps for which ΔG is significant.

Lysine acetyltransferases

Enzymes that catalyze the transfer of the acetyl group of acetyl-CoA to the ε-amino groups of lysyl residues, forming N-acetyl lysine (including histones)

Rate-limiting enzymes

Enzymes that regulate the rate of a metabolic pathway - Preferred targets of regulatory control

Deacetylases (2)

Enzymes that reverse the acetylation of certain proteins *(1) Histone deacetylases*: catalyze the removal by hydrolysis of acetyl groups, regenerating the unmodified form of the protein and acetate as products - Sirtuins: use NAD+ as substrate, which yields O-acetyl ADP-ribose and nicotinamide as products in addition to the unmodified protein

Allosteric enzymes

Have both an active site for substrate binding and an allosteric site for binding of an allosteric effector (activator, inhibitor) - Those for which catalysis at the active site may be modulated by the presence of effectors at an allosteric site - In general, binding of an allosteric regulator influences catalysis by inducing a conformational change that encompasses the active site

Regenerates the unmodified form of the protein and acetate as products

Histone deacetylases

Trans-acting regulatory proteins

Identify and bind target regulatory sequences on any chromosome

Feedback regulation

In some cases, the end product of a multistep biosynthetic pathway binds to and inhibits an enzyme catalyzing one of the early steps in that pathway. - In most cases, feedback inhibitors bind to the enzyme that catalyzes the first committed step in a particular biosynthetic sequence - The kinetics of feedback inhibition may be competitive, noncompetitive, partially competitive, or mixed

Where does protein degradation occurs?

In the interior of the proteasome (of cylindrical shape), specifically in the *26S proteasome*

Proenzymes

Inactive form of enzymes; enzymes must be activated to perform their catalytic function - Proteases are synthesized as catalytically inactive to protect tissues from their degradative effects - Selective, or "partial," proteolysis of a proprotein by one or more successive proteolytic "clips" converts it to a form that exhibits the characteristic activity of the mature protein, eg. its catalytic activity. - The proprotein forms of enzymes are termed *proenzymes or zymogens* - Proteins synthesized as proproteins include: the hormone insulin (proprotein = proinsulin), the digestive enzymes pepsin, trypsin, and chymotrypsin (proproteins = pepsinogen, trypsinogen, and chymotrypsinogen, respectively), several factors of the blood clotting and complement cascades, and the connective tissue protein collagen (proprotein = procollagen). - Proteolytic activation of proproteins is irreversible (one way) because reunification of the two portions of a protein produced by hydrolysis of a peptide bond is entropically disfavored. Once a proprotein is activated, it will continue to carry out its catalytic/other functions until it is removed by degradation or some other means - Found in GI tract and blood - Facilitate rapid mobilization of an activity in response to physiologic demand

Ideal enzyme for regulatory intervention

One whose quantity or catalytic efficiency dictates that the reaction it catalyzes is slow relative to all others in the pathway - Active control of homeostasis is achieved by the regulation of only a select subset of enzymes - Decreasing the catalytic efficiency or the quantity of the catalyst participating in the "bottleneck" or rate-limiting reaction will immediately reduce metabolite flux through the entire pathway, and vice versa

Covalent modifications

Phosphorylation, acetylation, and ubiquitination - Regulators of enzyme activity, protein degradation, etc. Provide a protein-based code for the storage and transmission of information cleavage of peptides -addition of phosphate groups -changes configuration of enzyme - may activate or inactivate

Potent agents for modifying protein structure and function.

Phosphorylation-dephosphorylation of proteins - Such ability lies in the *chemical properties of the phosphoryl group itself*. In order to alter an enzyme's functional properties, any modification of its chemical structure must influence the protein's three-dimensional configuration. - Have *high density of protein-bound phosphoryl groups*, generally -2 at physiologic pH - Have the propensity to form *strong salt bridges* with arginyl and lysyl residues - Have high exceptional hydrogen-bonding capacity

Cis elements

Regions of non-coding DNA which regulate the transcription of nearby genes

Regulation of enzyme quantity

Regulated by the product of the concentration of enzyme molecules and their intrinsic catalytic efficiency. Therefore catalytic capacity can be controlled by changing the *quantity of enzyme present*, *altering its intrinsic catalytic efficiency*, *or a combination thereof* - Induction of protein synthesis is a long process that takes hours to produce, whereas changes in intrinsic catalytic efficiency triggered by binding of *dissociable ligands (allosteric regulation)* or by *covalent modification* occur within fractions of seconds. Consequently, changes in protein level generally dominate when meeting long-term adaptive requirements, whereas changes in catalytic efficiency are favored for rapid and transient alterations in metabolite flux

Passive regulation of metabolite flow

Responds to changes in substrate level for coordination metabolite flow

Ubiquitin-proteasome pathway

Responsible both for the (1) regulated degradation of selected cellular proteins (eg. cyclins) and for (2) the removal of defective or aberrant protein species - Versatility and selectivity of the ubiquitin-proteasome system resides in the variety of *intracellular E3 ligases* and their ability to discriminate between the different physical or conformational states of target proteins - Selectively degrades proteins poor physical integrity and functional competency as a result of loss or damage to a prosthetic group, oxidation of cysteine or histidine residues, partial unfolding, or deamidation of asparagine or glutamine residues

Deacetylase enzyme that uses NAD+ as substrate

Sirtuins - Uses NAD+ as substrate, which yields O-acetyl ADP-ribose and nicotinamide as products in addition to the unmodified protein

Ubiquitin-proteasome pathway dysfunctions

Sometimes contribute to the accumulation and subsequent aggregation of misfolded proteins characteristic of several *neurodegenerative diseases*

Metabolite flow

Tends to be unidirectional. The flux of metabolites through metabolic pathways involves catalysis by numerous enzymes - Mean concentrations of metabolic intermediates remain relatively constant over time - While all chemical reactions are to some extent reversible, in living cells the *reaction products of one enzyme- catalyzed reaction serve as substrates for, and are removed by, other enzyme-catalyzed reactions*. As a result, many nominally reversible reactions occur unidirectionally - Covalent modifications regulate it (phosphorylation-dephosphorylation and acetylation- deacetylation)

Control of enzyme degradation

The *ubiquitin-proteasome pathway* degrades many proteins in animals - Degradation takes place in the *26S proteasome*, a large macromolecular complex made up of more than 30 polypeptide subunits arranged in the form of a hollow cylinder. The active sites of its proteolytic subunits face the interior of the cylinder, thus preventing indiscriminate degradation of cellular proteins. - *Proteins are targeted to the interior of the proteasome* (cylinder) by the covalent attachment of one or more molecules of ubiquitin, a small protein that is highly conserved among eukaryotes - "Ubiquination" is catalyzed by a large family of enzymes called *E3 ligases*, which attach ubiquitin to the side-chain amino group of lysyl residues on their targets - Recognition by proteolytic enzymes can be regulated by covalent modifications such as phosphorylation; binding of substrates or allosteric effectors; or association with membranes, oligonucleotides, or other proteins

Protein turnover.

The continuous breakdown and synthesis of body proteins involving the recycling of amino acids. - Even *constitutive* proteins, those whose aggregate concentrations remain essentially constant over time, are subject to continual turnover - Affected by hormonal, dietary, pathologic, and other factors

Regeneration of an unmodified protein is accomplished through __________ ?

The hydrolytic removal of phosphoryl groups from the molecule, which is catalized by the enzyme phoshatase

Active regulation of metabolite flow

The mechanisms that regulate enzyme efficiency in response to internal and external signals

Versatility of phosphorylation

The most common protein function affected is an *enzyme's catalytic efficiency*, phosphorylation can also alter its *location within the cell, susceptibility to proteolytic degradation, or responsiveness to regulation by allosteric ligands* - While phosphorylation of some increases their catalytic activity, the phosphorylated form of other enzymes may be catalytically inactive - Many protein kinases and most protein phosphatases act on more than one protein and are themselves interconverted between active and inactive forms by the binding of second messengers or by covalent modification by phosphorylation-dephosphorylation - The ability of many protein kinases and protein phosphatases to target more than one protein provides a means for an environmental signal to coordinately regulate multiple metabolic processes.

Allosteric effectors regulation of enzymes

The properties of the enzyme-effector complex differ from those of the separated enzyme and effector. - In some cases, the end product of a multistep biosynthetic pathway binds to and inhibits an enzyme catalyzing one of the early steps in that pathway, a process referred to as feedback regulation

Rate-limiting step

The slowest step in a metabolic pathway or series of chemical reactions, which determines the overall rate of the other reactions in the pathway.

Homeostasis

Walter Cannon coined the term "homeostasis" to describe the ability of animals to maintain a constant internal environment despite changes in their external surroundings. At the cellular level, homeostasis is maintained by adjusting the rates of key metabolic reactions in response to internal changes - Examples include the levels of key metabolic intermediates such as 5′-AMP and NAD+, or external factors such as hormones acting through receptor-controlled signal transduction cascades.

Can protein phosphorylation, acetylation, and other covalent modifications be considered another form of allosteric site?

Yes - Phosphorylation-dephosphorylation, acetylation-deacetylation, and feedback inhibition provide short-term, readily reversible regulation of metabolite flow in response to specific physiologic signals