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Chapt. 9 Regulation of Enzymes. Regulation of Enzymes Student Learning Outcomes : Explain that enzyme activities must be regulated for proper body function Explain three general mechanisms: Reversible binding in active site: substrate, inhibitors

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chapt 9 regulation of enzymes
Chapt. 9 Regulation of Enzymes
  • Regulation of Enzymes
  • Student Learning Outcomes:
  • Explain that enzyme activities must be regulated for proper body function
  • Explain three general mechanisms:
    • Reversible binding in active site:
      • substrate, inhibitors
    • Changing conformation of active site of enzyme:
      • Allosteric effectors, covalent modification,
      • Protein-protein interactions, zymogen cleavage
    • (Changing concentration of enzyme)
      • Synthesis, degradation
regulation of metabolic pathways
Regulation of metabolic pathways
  • Metabolic pathway analogous to cars on highway:
  • Flux of substrates affected by rate-limiting enzyme (barrier)
  • Removal of barrier increases flow
  • Activating rate-limiting enzyme

Fig. 9.1

regulation of glucose metabolism pathway
Regulation of glucose metabolism pathway
  • Ex. Regulation of glucose metabolism pathway:
  • Hexokinase & glucokinases convert glucose -> G-6-P in cells
  • Glycolysis for energy
    • Feedback regulation by ATP
  • Store G-6-P as glycogen
    • Feedforward by insulin
ii regulation by substrate product concentration
II. Regulation by substrate, product concentration
  • Michaelis-Menten equation describes kinetics:
  • More substrate gives more reaction, to maximal
  • Vi (initial velocity) relates to concentration of substrate [S] to Vmax (maximal velocity) and Km ([S] for 1/2 Vmax
  • Applies to simple reactions:
  • E + S  ES  E + P; k1 = forward, k2 back; k3 for E+P
  • Vi = Vmax[S]/ Km + [S] Km = k2 + k3/k1; Vmax = k3[Et]
ii regulation by substrate product concentration1
II. Regulation by substrate, product concentration:
  • Ex.Graph of Michaelis-Menten equation has limit of Vmax at infinite substrate.
  • Km = [S] where Vmax/2
  • Ex. Glucokinase Km 5 mM:
  • If blood glucose 4 mM ->
  • Vi = 0.44 Vmax
  • (Vm x 4mM/ (5mM + 4 mM)
  • Blood glucose 20 mM ->
  • Vi = 0.8 Vmax
  • (Vm x 20mM/ 5 + 20 mM

Fig. 9.2

different isozymes have different km for glucose
Different isozymes have different Km for glucose
  • Different hexokinases differ in Km for glucose:
  • glucose + ATP -> G-6-P + ADP
  • Hexokinase I
  • (rbc) only glycolysis
  • Glucokinase
  • (liver, pancreas) storage
  • Fasting blood sugar
  • about 5 mM (90 mg/dL) so
  • rbc can function even if low
  • blood sugar of glucose
  • S0.5 = half-max for S-shape curve

Fig. 9.3

reversible inhibitors decrease reaction velocity
Reversible inhibitors decrease reaction velocity
  • Regulation through active site: reversible inhibitors
  • Competitive inhibitors compete with substrate

Overcome by excess substrate (increase apparent Km)

  • Noncompetitive do not compete with substrate

Not overcome by substrate (lowers [E] and Vmax)

Products can also inhibit enzyme activity

Fig. 9.4

iii regulation through conformational changes
III. Regulation through conformational changes
  • Regulation through conformational changes of enzyme can affect catalytic site:
  • Allostery
    • – ex. Glycogen phosphorylase
  • Phosphorylation
    • – ex. Glycogen phosphorylase kinase
  • Protein-protein interactions
    • - ex. Protein kinase A
  • Proteolytic cleavage
    • - ex. chymotrypsinogen
a allosteric activators and inhibitors
A. Allosteric Activators and inhibitors
  • Allosteric enzymes:
  • Often multimeric,
  • Exhibit positive cooperativity in substrate binding (ex. Hemoglobin and O2)
  • T (taut state) inactive without substrate
  • R (relaxed) state active with substrate

Fig. 9.5

allosteric activators and inhibitors
Allosteric activators and inhibitors
  • Allosteric enzymes often cooperative S binding
  • Allosteric activators and inhibitors:
  • Bind at allosteric site,
  • not catalytic site
  • Conformational change
  • Activators often bind
  • R (relaxed) state
  • decrease S0.5
  • Inhibitors often bind
  • T (taut state)
  • increase S0.5

Fig. 9.6

b conformational change by covalent modification
B. Conformational change by covalent modification
  • Phosphorylation can activate or inhibit enzymes:
  • Protein kinases add phosphate
  • Protein phosphatases remove
  • PO42- adds bulky group,
  • negative charge, interacts
  • with other amino acids

Fig. 9.7

muscle glycogen phosphorylase regulation
Muscle glycogen phosphorylase regulation
  • Muscle glycogen phosphorylase is regulated by both phosphorylation and/or allostery:
  • Rate-limiting step glycogen -> glucose-1-PO4
  • ATP use increases AMP - allostery
  • phosphorylation increases activity
    • Signal from PKA

Fig. 9.8

ex protein kinase a
Ex. Protein kinase A
  • Protein kinase A: Regulatory, catalytic subunits:
  • Ser/thr protein kinase, phosphorylates many enzymes
    • Including glycogen phosphorylase kinase
  • Adrenline increase cAMP, dissociates R subunits,
    • Starts PO4 cascade

Fig. 9.9 cAMP activates PKA

other covalent modifications affect proteins
Other covalent modifications affect proteins
  • Covalent modifications affect protein activity, location in cell:
  • acetyl-

(on histones)

  • ADP-ribosylation
  • (as by cholera toxin on Ga subunit)
  • Lipid addition

(as on Ras protein)

Fig. 6.13 modified amino acids

conformational changes from protein protein interactions
Conformational changes from Protein-Protein interactions
  • Ca-Calmodulinfamily of modulator proteins
  • activated when [Ca2+ ] increases.
  • Ca2+/calmodulin binds to targets
  • e.g. protein kinases, allosteric result

CaMkinasefamily activated by Ca2+/calmodulin; phosphorylate metabolic enzymes, ion channels, transcription factors, regulate synthesis, release of neurotransmitters.

Fig. 9.10

small monomeric g proteins
Small monomeric G proteins
  • Small (monomeric) G proteins
  • affect conformation of other proteins:
  • GTP bound form binds and activates or inhibits
  • GDP bound form inactive
  • Other intermediates regulate the G proteins (GEF, GAP, etc)
  • Ras family (Ras, Rho, Rab, Ran, Arf)
  • diverse roles in cells

Fig. 9.11

proteolytic cleavage is irreversible
Proteolytic cleavage is irreversible
  • Proteolytic cleavage is irreversible conformational change:
  • Some during synthesis and processing
  • Others after secretion:
  • Proenzymes inactive:
    • Ex. Precursor protease is zymogen:
    • (chymotrypsinogen is cleaved by trypsin in intestine)
    • Ex. Blood clotting factors fibrinogen, prothrombin
regulation of pathways
Regulation of pathways
  • Regulation of metabolic pathways is complex:
  • Sequential steps, different enzymes, rate-limiting one
  • Match regulation to function of path

Fig. 9.12

lineweaver burk plot
Lineweaver-Burk plot
  • Lineweaver-Burk transformation converts Michaelis-Menten to straight line (y = mx + b)
  • double reciprocal plot
  • Ease of determining
  • Km and Vmax

Fig. 9.13

lineaver burk plots permit comparisons
Lineaver-Burk plots permit comparisons
  • Lineweaver-Burk plots permit analysis of enzyme kinetics, characterization of inhibitors

Fig. 9.14

key concepts
Key concepts
  • Key concepts:
  • Enzyme activity is regulated to reflect physiological state
  • Rate of enzyme reaction depends on concentration of substrate, enzyme
  • Allosteric activators or inhibitors bind sites other than the active site: conformational
  • Mechanisms of regulation of enzyme activity include: feedback inhibition, covalent modifications, interactions of modulator proteins (rate synthesis, degradation)
review questions
Review questions
  • 3. Methanol (CH3OH) is converted by alcohol dehydrogenases (ADH) to formaldehyde (CH2O), a highly toxic compound . Patients ingested toxic levels of methanol can be treated with ethanol (CH3CH2OH) to inhibit methanol oxidation by ADH. Which is the best rationale for this treatment?
  • Ethanol is structural analog of methanol – noncompetitive inhibitor
  • Ethanol is structural analog of methanol – will compete with methanol for binding enzyme
  • Ethanol will alter the Vmax of ADH for oxidation of methanol.
  • Ethanol is effective inhibitor of methanol oxidation regardless of the concentration of methanol
  • Ethanol will inhibit enzyme by binding the formadehyde-binding site on the enzyme, even though it cannot bind the substrate binding site for methanol.
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