Chapt. 9 Regulation of Enzymes

Regulation of EnzymesStudent 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

Fig. 9.1

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

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

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 concentration:

Fig. 9.2

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

Different isozymes have different Km for glucose

Fig. 9.3

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

Reversible inhibitors decrease reaction velocity

Regulation through active site: reversible inhibitors

A.Competitive inhibitors compete with substrateOvercome by excess substrate (increase apparent Km)

B.Noncompetitive do not compete with substrateNot overcome by substrate (lowers [E] and Vmax)

Fig. 9.4

Products can also inhibit enzyme activity

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

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

Fig. 9.6

Allosteric enzymes often cooperative S bindingAllosteric 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

B. Conformational change by covalent modification

Fig. 9.7

Phosphorylation can activate or inhibit enzymes:

Protein kinases add phosphate

Protein phosphatases remove

• PO42- adds bulky group,

negative charge, interacts with other amino acids

Muscle glycogen phosphorylase regulation

Fig. 9.8

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

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

Covalent modifications affect protein activity, location in cell:

• acetyl- (on histones)

• ADP-ribosylation (as by cholera toxin on

G subunit)

• Lipid addition(as on Ras protein)

Fig. 6.13 modified amino acids

Conformational changes from Protein-Protein interactions

Fig. 9.10

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

Ca-Calmodulin family of modulator proteins• activated when [Ca2+ ] increases. • Ca2+/calmodulin binds to targets

e.g. protein kinases, allosteric result

Small monomeric G proteins

Small (monomeric) G proteinsaffect 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 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 metabolic pathways is complex:

Sequential steps, different enzymes, rate-limiting one

Match regulation to function of path

Fig. 9.12

Lineweaver-Burk plot

Fig. 9.13

Lineweaver-Burk transformation converts Michaelis-Menten to straight line (y = mx + b)

• double reciprocal plot

• Ease of determining Km and Vmax

Lineaver-Burk plots permit comparisons

Lineweaver-Burk plots permit analysis of enzyme kinetics, characterization of inhibitors

Fig. 9.14

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

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?

a.Ethanol is structural analog of methanol – noncompetitive inhibitor

b.Ethanol is structural analog of methanol – will compete with methanol for binding enzyme

c.Ethanol will alter the Vmax of ADH for oxidation of methanol.d.Ethanol is effective inhibitor of methanol oxidation regardless

of the concentration of methanole.Ethanol will inhibit enzyme by binding the formadehyde-

binding site on the enzyme, even though it cannot bind the substrate binding site for methanol.