6.5 Regulatory Enzymes · Biochemistry 2027

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

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

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

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

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

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

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

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

References

**표 6.5-1**Regulatory mechanisms compared
Mechanism	Chemical change	Example	Reversibility and character
Allosteric control	Non-covalent effector binding	CTP on ATCase	State equilibrium shifts with binding
Covalent phosphorylation	Phosphate ester on Ser, Thr or Tyr	Glycogen metabolism enzymes	Kinase and phosphatase act in opposition
Multiple phosphorylation	Combinations across several sites	MAPK signalling proteins	Can tune threshold and timing but is not obligatory
Proteolytic activation	Cleavage of the peptide backbone	Trypsinogen	Direct reversal of cleavage is not usual
Cascade	Stepwise zymogen activation	Tissue factor, thrombin, fibrin	Amplification and restraint are both required
Multiple input integration	Combinations of the above	PFK-1, glycogen breakdown	Depends on cell and tissue context