BIOCHEM2027 Specialised Subject · 4.4
2027 · High School Specialised Subject · Biochemistry

6.5 Regulatory Enzymes

Regulatory enzymes

Allosteric control and cooperativity, Hill-type kinetics, reversible phosphorylation, multiple phosphorylation, zymogen activation, the clotting cascade and layered regulation.

8 SECTIONS22 FIGURESEnglish edition
01

Allosteric enzymes change conformation on effector binding

  • Allosteric control means binding at one site changes structure or function at another
  • The effector site is often distinct from the active site, and an effector that raises activity is an activator while one that lowers it is an inhibitor
  • The T and R state model is a simplification and real enzymes may occupy more states
  • Effectors shift the population by changing the relative stability of particular states
  • Cooperativity means binding at one site changes affinity or reactivity at another, though not every allosteric enzyme is cooperative
  • Example: ATCase receives negative feedback from CTP and a balancing signal from ATP
Figure 6.5-83Main molecular pathways through which regulatory enzymes act
Figure 6.5-84Conceptual shift in the protein state equilibrium on effector binding
Figure 6.5-89Distinguishing allosteric regulation from substrate cooperativity
Figure 6.5-88Metabolic feedback control of ATCase and the roles of ATP and CTP
02

Kinetics of regulatory enzymes can depart from Michaelis-Menten behaviour

  • Hill-type approximation for positive Cooperativity: v/Vmax = [S]^nH/(K0.5^nH + [S]^nH)
  • K0.5 is the substrate concentration giving half Vmax, and nH measures steepness rather than the number of sites
  • nH of 1 gives a hyperbola, above 1 a sigmoid, and below 1 can reflect negative cooperativity or other heterogeneity
  • K-type effectors shift apparent K0.5, V-type effectors change maximum rate, and both can change together
  • Example: K0.5 2.0 mM, nH 2 and Vmax 100 give v = 80 at 4.0 mM, and 90 percent is reached at 6.0 mM
  • The Hill plot uses log{v/(Vmax - v)} against log[S], and real slopes are not constant across all concentrations
Figure 6.5-85Michaelis-Menten and Hill-type sigmoid curves compared (model)
Figure 6.5-86K-type curve shift produced by an allosteric effector (model)
Figure 6.5-87Hill plot and interpretation of its slope (teaching model)
03

Some enzymes are controlled by reversible covalent modification

  • Protein kinases transfer the ATP phosphate to Ser, Thr or Tyr hydroxyls, and phosphatases hydrolytically remove it
  • Phosphorylation adds negative charge and affects hydrogen bonds, salt bridges, metal binding and protein interactions
  • It activates some enzymes and inhibits others, so phosphorylation always means activation is not a rule
  • Glycogen phosphorylase is activated by phosphorylation while glycogen synthase is inhibited, preventing futile simultaneity
  • Simple model df/dt = kP(1 - f) - kD f gives f* = kP/(kP + kD), for example 0.75 and a 2.5 minute time constant with kP 0.30 and kD 0.10
Figure 6.5-90Reversible control of protein function by phosphorylation and dephosphorylation
Figure 6.5-91Metabolic enzymes in which phosphorylation has opposite effects
Figure 6.5-92Fractional change with the balance of phosphorylation and dephosphorylation (model)
04

Multiple phosphorylation tunes activity precisely

  • Several Ser, Thr or Tyr sites allow multiple phosphoforms; two independent sites give the four states 00, 10, 01 and 11
  • Which form is active depends on the protein and the cellular context
  • Under independence the fractions with 0, 1 and 2 phosphates are (1-p) squared, 2p(1-p) and p squared, so p = 2/3 gives 4/9 for both sites
  • Processive phosphorylation modifies several sites in one binding event, distributive phosphorylation requires rebinding each time
  • Repeated binding and phosphatase competition can create threshold responses, though not every multi-site system is ultrasensitive
Figure 6.5-93State distribution of two independent phosphorylation sites (teaching probability model)
Figure 6.5-94Processive and distributive phosphorylation compared
Figure 6.5-102Gradual and switch-like responses expressed in a Hill model
05

Some enzymes are activated by proteolytic cleavage of an inactive precursor

  • A zymogen is a catalytically suppressed precursor that becomes active when a specific peptide bond is cleaved
  • Digestive enzymes are secreted as trypsinogen and chymotrypsinogen and activated in the duodenum, limiting self-digestion
  • Enteropeptidase activates trypsinogen first and the resulting trypsin activates the other zymogens
  • Because a backbone bond is cut, this control is not spontaneously reversed as phosphorylation is, and inhibitor binding or removal ends the action
Figure 6.5-95Activation of digestive zymogens and the principle of cascade proteolysis
Figure 6.5-96Chemical difference between reversible phosphorylation and proteolytic activation
06

A cascade of proteolytically activated zymogens produces clotting

  • Tissue factor exposed by injury lets TF-VIIa activate factor X, and Xa with Va, Ca2+ and phospholipid converts prothrombin to thrombin
  • Thrombin cleaves fibrinogen to fibrin and XIIIa cross-links it to stabilise the clot
  • The classical intrinsic and extrinsic schemes are teaching aids; clotting in vivo is initiation, amplification and propagation regulated on cell membranes and platelets
  • Factors V and VIII are mainly cofactors and XIII is a transglutaminase, so not every clotting factor is a protease
  • The cascade amplifies signal while TFPI, antithrombin and the protein C and S system restrain it, and normal haemostasis is that balance
Figure 6.5-97Core clotting pathway from tissue factor to fibrin formation
Figure 6.5-98Normalised model of signal amplification in the clotting cascade
Figure 6.5-99Main negative regulatory pathways that restrain coagulation
07

Some regulatory enzymes combine several mechanisms

  • PFK-1 converts fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP, with ATP both a substrate and an allosteric inhibitor
  • AMP and ADP signal low energy and favour activity, fructose 2,6-bisphosphate is a strong activator and citrate can inhibit
  • Control of PFK-1 itself and phosphorylation of the bifunctional PFK-2/FBPase-2 that sets F2,6BP are different layers
  • The effect of PFK-2/FBPase-2 phosphorylation depends on tissue-specific isoforms, so no single generalisation holds
  • Glycogen phosphorylase phosphorolyses alpha(1-4) bonds with Pi to give glucose 1-phosphate, and hormonal signals change its phosphorylation through cAMP and PKA
  • When activity changes, examine initial-rate curves, effector binding, phosphorylation sites, cleavage state, localisation and actual metabolites together
Figure 6.5-100Integrated control of PFK-1 by substrates, activators and inhibitors
Figure 6.5-101Glycogen breakdown controlled by hormone signals combined with energy status
Figure 6.5-103Flow of evidence used to establish the molecular mechanism of a regulatory enzyme
08

Integrated comparison

  • Regulatory enzymes either change catalytic efficiency or change the fraction, timing and location of the active state
  • Allostery is fast and reversible through non-covalent binding, phosphorylation is covalent and intermediate in speed, and zymogen cleavage is irreversible and needs a separate termination route
  • Cascades gain amplification but require restraining systems for normal haemostasis
  • Observing a rate change does not establish a molecular mechanism, which needs evidence from several levels
표 6.5-1Regulatory mechanisms compared
Figure 6.5-104Five core principles of regulatory enzymes in unit 6
2027 High School Specialised Subject · Biochemistry · Sangil Park (Asan High School) · Reference material is printed as an appendix.
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