Chapt. 9 Regulation of Enzymes - PowerPoint PPT Presentation

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Chapt. 9 Regulation of Enzymes

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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 – PowerPoint PPT presentation

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Title: Chapt. 9 Regulation of Enzymes


1
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

2
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
3
Regulation of glucose metabolism pathway
  • Ex. Regulation of glucose metabolism pathway
  • Hexokinase glucokinases convert glucose -gt
    G-6-P in cells
  • Glycolysis for energy
  • Feedback regulation by ATP
  • Store G-6-P as glycogen
  • Feedforward by insulin

4
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 EP
  • Vi VmaxS/ Km S Km k2 k3/k1
    Vmax k3Et

5
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 -gt
  • Vi 0.44 Vmax
  • (Vm x 4mM/ (5mM 4 mM)
  • Blood glucose 20 mM -gt
  • Vi 0.8 Vmax
  • (Vm x 20mM/ 5 20 mM

Fig. 9.2
6
Different isozymes have different Km for glucose
  • Different hexokinases differ in Km for glucose
  • glucose ATP -gt 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
7
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
8
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

9
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
10
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
11
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
12
Muscle glycogen phosphorylase regulation
  • Muscle glycogen phosphorylase is regulated by
    both phosphorylation and/or allostery
  • Rate-limiting step glycogen -gt glucose-1-PO4
  • ATP use increases AMP - allostery
  • phosphorylation increases activity
  • Signal from PKA

Fig. 9.8
13
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
14
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
15
Conformational changes from Protein-Protein
interactions
  • Ca-Calmodulin family of modulator proteins
  • activated when Ca2 increases.
  • Ca2/calmodulin binds to targets
  • e.g. protein kinases, allosteric result

CaM kinase family activated by Ca2/calmodulin
phosphorylate metabolic enzymes, ion channels,
transcription factors, regulate synthesis,
release of neurotransmitters.
Fig. 9.10
16
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
17
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

18
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
19
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
20
Lineaver-Burk plots permit comparisons
  • Lineweaver-Burk plots permit analysis of enzyme
    kinetics, characterization of inhibitors

Fig. 9.14
21
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)

22
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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