Transcript Allosteric 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
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
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:
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 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
Regulation through active site: reversible inhibitors
A. Competitive inhibitors compete with substrate
Overcome by excess substrate (increase apparent Km)
B. 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
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
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
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 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
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
Ga subunit)
• Lipid addition
(as on Ras protein)
Fig. 6.13 modified amino acids
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
Fig. 9.10
CaM kinase family
activated by
Ca2+/calmodulin;
phosphorylate
metabolic enzymes,
ion channels,
transcription factors,
regulate synthesis,
release of
neurotransmitters.
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
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
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
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 methanol
e. Ethanol will inhibit enzyme by binding the formadehydebinding site on the enzyme, even though it cannot bind the
substrate binding site for methanol.