Unit 7 Question Bank
Note: your TMU slide 11 describes only the CTP inhibition; the ATP activation is an addition in the Harper's edition in your folder, not a contradiction.Harper's ch.9, p.91 — extends TMU Lecture 7 Slide 11
Origin of the term: Jacques Monod reasoned that because most feedback inhibitors bear no structural similarity to the substrate, they cannot be isosteric with it but must be allosteric — “occupying another space”. The existence of spatially distinct active and allosteric sites has since been confirmed by X-ray crystallography and site-directed mutagenesis.
Kinetics: in K-series enzymes the inhibitor raises Km without affecting Vmax; in V-series enzymes it lowers Vmax without affecting Km. The terms “competitive” and “non-competitive” should not be used, as they carry misleading mechanistic implications.Harper's ch.9, p.91 · TMU Lecture 7 Slides 8, 13
Inhibiting an early step, rather than the last one, prevents the useless accumulation of intermediates. In branched pathways, additional refinements provide fine control: multiple feedback loops regulating enzymes common to several end products, cooperative feedback inhibition (the effect of two or more end products being additive or greater), and multiple isoforms of one enzyme, each sensitive to a different end product, so that flux through the shared segment is reduced but not eliminated.
⚠ Distinguish from feedback regulation, a phenomenological term devoid of mechanistic implications.Harper's ch.9, pp.90–91 · TMU Lecture 7 Slide 14
Examples: pepsinogen → pepsin; trypsinogen → trypsin; chymotrypsinogen → chymotrypsin; proinsulin → insulin; procollagen → collagen; several clotting and complement factors.
Why: secretion in inactive form protects the tissue of origin from autodigestion (as in pancreatitis); it permits rapid mobilisation of activity in response to physiological need, for which induction of synthesis would be far too slow; and it is a simple and economical, albeit one-way, means of restraining latent activity.
Irreversible because cells cannot reunite the two portions of a protein produced by hydrolysis of a peptide bond.Harper's ch.9, pp.92–93 · TMU Lecture 7 Slide 21
By contrast, the concentrations of many enzymes depend on the presence of inducers — typically substrates or structurally related compounds — that initiate their synthesis. Induction of protein synthesis is a complex multistep process requiring hours, and therefore serves long-term adaptive requirements rather than rapid change.Harper's ch.9, pp.89–90 · TMU Lecture 7 Slide 7
Decreasing the quantity or catalytic efficiency of that enzyme immediately reduces flux through the entire pathway; increasing either enhances it. Such enzymes are therefore efficient targets for drugs: the statins inhibit HMG-CoA reductase, the rate-limiting enzyme of cholesterogenesis, and acetyl-CoA carboxylase catalyses the first committed reaction of fatty acid biosynthesis.Harper's ch.9, p.89
It targets proteins to the interior of the 26S proteasome, a complex of more than 30 subunits forming a hollow cylinder whose proteolytic active sites face inward, preventing indiscriminate degradation. The system degrades both regulated proteins (e.g. the cyclins) and proteins damaged by loss of a prosthetic group, oxidation of cysteine or histidine, or deamidation of asparagine or glutamine.Harper's ch.9, p.90
Protein kinases transfer the terminal γ-phosphoryl group of ATP to the hydroxyl groups of seryl, threonyl or tyrosyl residues; protein phosphatases remove it by hydrolysis, not by recombining phosphate with ADP. Both directions are thermodynamically favourable because they are two different reactions, not one reaction run in reverse.
Contrast the irreversible modification, partial proteolysis.Harper's ch.9, pp.92–93
The requirement
Homeostasis is the maintenance of a relatively constant intracellular and intra-organ environment despite wide fluctuations in the external environment, achieved by appropriate changes in the rates of biochemical reactions in response to physiological need. Regulation may be passive or active.
1 · Passive control — substrate concentrations near Km
Near Km the substrate-saturation curve is steep, so a given increment in [S] produces a large change in velocity; far above Km the curve has flattened and the same increment produces almost none. For most enzymes the average intracellular substrate concentration therefore tends to lie close to Km, placing the enzyme at maximum sensitivity so that changes in substrate concentration automatically generate corresponding changes in metabolite flux.
2 · Compartmentation
Anabolic and catabolic pathways that interconvert common products are separated into distinct subcellular compartments — fatty acid biosynthesis in the cytosol, fatty acid oxidation in mitochondria. Antagonistic pathways may also coexist without a physical barrier provided each proceeds via unique intermediates. A further, chemical form of separation is the discrimination between NAD⁺, reduced by enzymes generating electrons for the electron transport chain, and NADPH, the electron donor for reductive biosynthesis.
3 · The target of active control — the rate-limiting step
Although a pathway involves many enzymes, active control is exerted over only a select subset. The ideal target is the enzyme whose reaction is slow relative to all others: altering its quantity or catalytic efficiency immediately alters flux through the entire pathway. Examples are acetyl-CoA carboxylase, catalysing the first committed reaction of fatty acid synthesis, and HMG-CoA reductase, the rate-limiting enzyme of cholesterogenesis and the target of the statins.
4 · Regulating the QUANTITY of enzyme — long-term
- Control of synthesis. Constitutive enzymes are present at constant levels; the concentrations of others depend on inducers, typically substrates or related compounds, that initiate their synthesis.
- Control of degradation. Proteins are degraded by ATP- and ubiquitin-dependent pathways and by ATP-independent ones. E3 ligases attach ubiquitin to lysyl side chains, targeting the protein to the 26S proteasome.
Induction of protein synthesis requires hours, so changes in protein level suit long-term adaptive requirements.
5 · Regulating CATALYTIC EFFICIENCY — short-term
Changes in intrinsic catalytic efficiency are achieved within seconds, and are therefore suited to rapid, transient alterations in flux.
- Allosteric regulation. A small effector binds at a site spatially distinct from the catalytic site, inducing a conformational change that alters Km (K-series) or Vmax (V-series). Most feedback inhibition — the end product of a pathway inhibiting an early enzyme — operates this way, and many hormones act through allosteric second messengers.
- Reversible covalent modification. Protein kinases transfer the γ-phosphoryl group of ATP to seryl, threonyl or tyrosyl residues; protein phosphatases remove it hydrolytically. The ease of interconversion allows an enzyme's properties to be altered only for as long as the need persists.
- Irreversible covalent modification. Selective proteolysis of catalytically inactive proenzymes initiates conformational changes that form the active site — used where activity must be mobilised rapidly and once only, as in digestion and blood clotting.
Conclusion
The protein kinases and phosphatases participating in regulatory cascades that respond to hormonal and second-messenger signals constitute a “bio-organic computer” able to process and integrate complex environmental information and produce an appropriate, comprehensive cellular response.
Definition
Allosteric regulation is regulation in which a small molecule — an allosteric effector — binds at an allosteric site, spatially distinct from the catalytic site of the target enzyme, producing a change in the enzyme's intrinsic catalytic efficiency. Allosteric enzymes are accordingly those whose catalysis at the active site may be modulated by the presence of effectors at an allosteric site.
Origin of the concept
Jacques Monod proposed the existence of sites physically distinct from the catalytic site. His reasoning was structural: most feedback inhibitors bear no structural similarity to the substrate of the enzyme they regulate, so they cannot plausibly occupy the substrate's site. Such effectors are therefore not isosteric with the substrate but allosteric — “occupying another space”. The hypothesis has since been confirmed by X-ray crystallography and site-directed mutagenesis in a variety of enzymes.
The model enzyme — aspartate transcarbamoylase
ATCase catalyses the first reaction unique to pyrimidine biosynthesis and is feedback-inhibited by CTP, an end product of that pathway. The classic evidence: after treatment with mercurials, ATCase loses its sensitivity to CTP but retains full activity for the synthesis of carbamoyl aspartate. Since catalytic and regulatory functions can be separated, CTP must bind at a site different from the substrate. The enzyme consists of multiple catalytic and regulatory subunits, the latter binding the nucleotide triphosphates that modulate activity. Harper's adds that the purine nucleotide ATP activates the enzyme and can overcome CTP inhibition, balancing purine and pyrimidine synthesis.
Kinetic classes
Referring to allosteric inhibition as “competitive” or “non-competitive” carries misleading mechanistic implications. Two classes are recognised instead:
| Class | Effect | Probable structural basis |
|---|---|---|
| K-series | Km raised, Vmax unaffected | The conformational change weakens the bonds between substrate and the substrate-binding residues |
| V-series | Vmax lowered, Km unaffected | The change alters the orientation or charge of the catalytic residues |
In general, binding of an allosteric regulator influences catalysis by inducing a conformational change that encompasses the active site; intermediate effects on both constants may also be observed.
Physiological importance
Allosteric regulation is the mechanism by which most feedback inhibition operates, and many hormones act through allosteric second messengers. Because it depends only on the binding of a dissociable ligand, it takes effect within seconds, making it suited to rapid and transient alterations in metabolite flux.
Framing
Covalent modification alters an enzyme's intrinsic catalytic efficiency and, like allosteric regulation, acts within seconds — making it suited to rapid, transient control, as distinct from the hours required to change enzyme levels by induction of synthesis. Modifications fall into two classes with quite different physiological uses.
Irreversible modification — selective proteolysis
Certain proteins are synthesised as inactive precursors, proproteins; the proprotein forms of enzymes are called proenzymes or zymogens. Selective, or “partial”, proteolysis — one or more highly specific proteolytic clips — converts them to the active form.
Crucially, proteolysis often produces conformational changes that properly configure the active site. In α-chymotrypsin the catalytically essential His 57 and Asp 102 reside on the B peptide while Ser 195 resides on the C peptide; the conformational changes accompanying proteolysis of prochymotrypsin align the three residues of the charge-relay network — the Asp102–His57–Ser195 catalytic triad — thereby forming the catalytic site. The resulting three peptides remain associated by interchain disulfide bonds, demonstrating that contact and catalytic residues can lie on different chains yet still be within bond-forming distance of the substrate.
The modification is physiologically irreversible because cells cannot reunite the two portions of a protein produced by hydrolysis of a peptide bond; reunification is entropically disfavoured. Once activated, the protein acts until removed by degradation.
Why this suits its purpose. Secretion of proteases as inactive proenzymes protects the tissue of origin from autodigestion, as occurs in pancreatitis; and it permits rapid mobilisation of an activity in response to physiological demand — blood clot formation, clot dissolution and tissue repair are brought “on line” only when needed, and induction of synthesis would be far too slow to respond to something like the loss of blood. Examples: pepsinogen, trypsinogen, chymotrypsinogen, proinsulin, procollagen and the clotting and complement factors.
Reversible modification — phosphorylation and others
Acetylation, ADP-ribosylation, methylation and phosphorylation are all reversible — meaning that the modified protein can be restored to its original state, not that the same reaction runs backwards.
Phosphorylation-dephosphorylation is by far the commonest. Protein kinases catalyse transfer of the terminal γ-phosphoryl group of ATP to the hydroxyl groups of seryl, threonyl or tyrosyl residues, forming O-phosphoseryl, O-phosphothreonyl and O-phosphotyrosyl residues. Protein phosphatases regenerate the unmodified protein by hydrolytic removal of the phosphoryl group. A typical mammalian cell contains thousands of phosphorylated proteins and several hundred kinases and phosphatases.
The thermodynamic subtlety. If the reaction introducing a modification is favourable, simply reversing it would be rendered impractical by the correspondingly unfavourable free-energy change. Phosphorylation is favourable because it exploits the high-energy γ-phosphoryl group of ATP; the phosphate is therefore removed not by recombining it with ADP but by a hydrolytic reaction — a second, separately favourable process. The same logic applies to acetylation, where acetyltransferases use the high-energy donor NAD⁺ and deacetylases catalyse a direct hydrolysis yielding free acetate.
Why phosphorylation is used so widely. First, the ease of interconversion: an enzyme's functional properties can be altered only for as long as a specific need persists, then restored, poised for the next stimulus. Second, the chemical properties of the phosphoryl group itself, which is bulky and strongly charged and so substantially alters local structure.
A third category — permanent structural modification
Prenylation, glycosylation, hydroxylation and fatty acid acylation introduce features into newly synthesised proteins that persist for the lifetime of the protein. These are structural rather than regulatory.
The histone code
Histones and other chromatin proteins are extensively modified by acetylation, methylation, ADP-ribosylation and phosphorylation, altering how they interact with one another and with DNA — either rendering genes more accessible to the transcription machinery or silencing expression. This constitutes the “histone code”, a classic example of epigenetics: the hereditary transmission of information by a means other than the nucleotide sequence.