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 Explain three general mechanisms: Reversible binding in active site: substrate, inhibitors

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Chapt. 9 Regulation of Enzymes

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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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