LSM1101_Enzyme3

LSM1101 LECTURE 3 Enzyme Regulation/ Application

Dr Deng Lih Wen

Dept of Biochemistry

Michaelis-Menton Plot

E + S ES E + Pk1

k-1

k2

[ ][ ] S S

M

max0

+⋅

=KVV

vo

[S]Km

Vmax/2

Vmax

Equilibrium assumptionSteady state assumption

[S] < KM

[ ]S M

max0

KVV ≈

First order

[S] = KM

2/ max0 VV =

[S] >> KM

max0 VV =

Zero order

Double-Reciprocal Plot(Lineweaver-Burk Plot)

[ ][ ] S S

M

max0

+⋅

=KVV

[ ] 1

S1

1

maxmax

M

0 VVK

V+⎟

⎠⎞

⎜⎝⎛=

[ ][ ]S

S 1max

M

0 ⋅+

=VK

V

baXY +=

CompetitiveChanges Km

Uncompetitive Both Km and Vmax decrease

Mixed Changes Vmax, may not affect Km

EnzymeDNA transcription

mRNA translation Amino acidturnover

turnover

nucleotide Allosteric regulation Covalent modification

Proteolytic cleavage Compartmentation

Control of enzyme synthesis amounts Control of enzyme activity

Regulation of Overal Enzyme Activity

Hexokinase: 4 isoenzymes• Hexokinase I, II, III: most of tissues except liver, broad substrate specificity• Hexokinase IV (also called Glucokinase): predominate in liver

Phosphorylation of Glucose

Isoenzymes (Isozymes): catalyze the same chemical reaction but are encoded by different genes. Isoenzymes might be expressed at different tissues and exhibit different regulatory properties.

Hexokinase &Glucokinase

Hexokinase (I, II, III)Most of tissues except liverKm for glucose ~0.1mMAllow for utilization of glucose even when blood glucose is low (such as brain)

Glucokinase (Hexokinase IV)LiverKm for glucose ~10mMBuffering effect on blood glucose

[Glucose] (after meal)• excess glucose transported to hepatocytes and converted into G6P• GK exhibit high Km, thus, its activity continues to increase as the glucose concentration rises to 10mM or more

[Glucose] (fasting)• F6P triggers the association of

GK and GKRP• complex in the nucleus, inactive• Liver does not compete with other organs for the limited glucose.

Glucokinase in HepatocyteHepatocyte (liver cell)

Fig 8.14, Lippincott’s illustrated Reviews in Biochemistry

Kinetics property (high Km)Localization (GK-GKRP in nucleus)Transcriptional regulation by insulin

blood glucose level rise

Increase insulin release (β-cells of pancreas)

Insulin promotes transcription of the glucokinase gene, resulting in an increase in liver glucokinase amounts

About half of the newly secreted insulin is extracted by the liver

* Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days)

Ways to regulate glucokinase activity

Allosteric EnzymesAllosteric is derived from the Greek root “allo”, meaning “the other”.

Regulated by molecules called effectors (or modulators) that bind noncovalently at a site that other than the active site.

Composed of multi-subunits.

Presence of effectors (modulators) alter enzyme activityNegative effectors – inhibitionPositive effectors – activation

Homotropic effectors – substrate serves as a modulatorHeterotropic effectors – modulator is different from the substrate

Allosteric Enzymes Undergo Conformational Changes in Response to Modulator Binding

Principles of Biochemistry 4th Edition, Fig 6-26

Binding of the positive modulator (M) to its specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change.

Aspartate Transcarbamoylase

- modulator + modulator

Feedback Inhibition

In many pathways, a regulated step is catalyzed by an allostericenzymeBuilding up of the end product ultimately slows the entire pathway.The conversion of L-threonine to L-isoleucine in five steps is heterotropic allosteric feed-back inhibitionProduct inhibition

Principles of Biochemistry 4th Edition, Fig 6-28

Allosteric Regulation Kinetics Profiles• Do not exhibit typical MM hyperbolic curve• If substrate is a homotropic effector, a cooperative sigmoidcurve is observed.(binding of substrate to one active site makes it easier for additional substrate molecule to bind to the other sites of the multi-meric enzyme.

negative effector

positive effector

K0.5(In the above figure, "normal" means an enzyme that does NOT show cooperative substrate binding; it could be monomeric, or a multimeric enzyme with no communication between binding sites.)

Feedback inhibition of ATCaseregulates Pyrimidine Synthesis

Negative modulator: CTP (feedback inhibition)Positive modulator: ATP• When ATP > CTP, ATCase is activated to synthesize pyrimidine nucleotides until the conc of ATP and CTP become balanced.

• Coordinate the rates of synthesis of purineand pyrimidine nucleotides, which are required in roughly equal amounts in nucleic acid synthesis.

inhibition

Binds cooperatively to ATCase

Regulation of Enzyme Activity

DNA mRNA Enzyme Amino acidtranscription translation

turnover

turnover



Zymogens (Proenzymes)

Inactive enzyme precursor; cleavage to be activatedExamples:

Hormones Proteolytic enzymes of the digestive tractBlood clotting

The mature hormone insulin consists of the disulfide-linked A and B chains

CarboxypeptidaseProcarboxypeptidasePancreasElastaseProelastasePancreasPepsinPepsinogenStomachActive ProteaseZymogenOrigin

TrypsinTrypsinogenPancreasChymotrypsinChymotrypsinogenPancreas

Pancreatic and Gastric Zymogens

Fig 19.4, Lippincott’s illustrated Reviews in Biochemistry

Cleavage of dietary protein by proteases

• Removal of the N-terminal hexapeptide is catalyzed by either enteropeptidase or trypsin(autocatalysis)

The Proteolytic Activation of Chymotrypsinogen

Biochemistry 3rd Ed, Figure 15.3

Blood Clotting, the result of a series of zymogen activition

•Activation of thrombin promotes the conversion of fibrinogen into fibrin aggregates into ordered filamentous form the clot.


• Cascade of enzymatic activation allows blood clotting to occur rapidly in response to injury

•The intrinsic and extrinsic pathways converge at Factor Xa.

Regulation of Enzyme Activity

DNA mRNA Enzyme Amino acidtranscription translation

turnover

turnover



Covalent Modification

Phosphorylation/Dephosphorylation

Principles of Biochemistry

Phosphorylation of a Ser (or a Thr or a Tyr) residue of enzyme to make a phosphate ester modification such modification changes the enzyme activity.

• Modifying enzyme is a protein kinase. (Some are specific for a specific protein, others are more promiscuous -- phosphorylate a variety of protein substrates.) • "DEmodifying" enzyme is a phosphoprotein phosphatase.• Phosphorylation of enzyme can be MORE active, but that depends on the specific target enzyme; some target enzymes are LESS active as a result of phosphorylation.

Regulation of glycogen phosphorylaseactivity by phosphorylation

(Glucose)n + Pi (glucose)n-1 + glucose 1-phosphateGlycogen

glucose 6-phosphate

Convert to glucose

ATP synthesis

muscleliver

Breakdown of glycogen in muscles and liver is regulated by variations in the ratio of a and b form.

Glycogen phosphorylase

phosphoglucomutase

Glycolysis

Export to other tissues

(Glucose)n + Pi (glucose)n-1 + glucose 1-phosphateGlycogen phosphorylase

Glycogen Phosphorylase(Allosteric Regulation and Covalent Modification)

P

Pinactive active

Covalent control

active

Allo

ster

icco

ntro

l


AMP G-6-P,ATP

ATP and G6P: negative heterotropic effectorsAMP: a positive heterotrophic effector

Industrial Application

Food industryfermented alcohol drinks, lactose free milk, animal feed

Cleaning compoundsLaundry detergents, color brightening and softening

Production of Antibiotics from microorganisms

Industrial Enzymes for Food Industry

Fermented alcohol drinks, beer brewing Glucanase: release starch from endospermAmylase : breakdown starch to sugars (glucose)Peptidase: hydrolyze proteins to amino acids

• Lactose intolerance: inability to metabolize lactose due to low lactase activity. • Lactose-free milk can be produced by passing milk over lactase enzyme bound to an inert carrier; once the molecule is cleaved, there are no lactose ill-effects.

Lactose free milk

• Enzyme supplementation: cellulase, hemicellulase(including β-glucanase and xylanase), amylase, and phytase.• Increase feed utilization and digestion by catalyzing the breakdown of nonstarch polysaccharides

Animal Feed

Industrial Enzymes for Detergents

Many classes of enzymes are known to improve the laundry process:

Proteases remove protein stains such as grass, blood, egg and human sweat.

Amylases remove residues of starch-based foods like potatoes, spaghetti, custards, gravies and chocolate.

Lipases are effective in removing oil / greasy body and food stains

Cellulases

provide general cleaning benefits, especially on dust and mud

Color brightening and softening. http://www.scienceinthebox.com/en_UK/glossary/enzymes2_en.html

Production of Antibiotics-Penicillin

"On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to Their Use in the Isolation of B. Influenzas“ The British Journal of Experimental Pathology, (1929) x: p.226.

Dr Alexander Fleming

Clinical Applications

Diagnostic tools Analytical toolsTherapeutic tools

An important tool in diagnosis and monitoring of treatment.

These quantities increase in some diseases of tissues and organs, since as a consequence of increased death cells or changes in cell membranes permeability, intracellular enzymes are released into plasma, giving clues about some organs diseases.

Measuring Plasma Enzyme Activity

Kinetics of release of cardiac enzymes into serum following a myocardial infarction

Creatine Kinase

* 3 isoenzymes, each of them composed of two polypeptides (called B and M subunit)

CK1 = BB, CK2 = BM, CK3 = MMSkeletal muscle: 98% CK3 and 2% CK2Cardiac muscle: 70% CK3 and 30% CK2Other tissues: Mainly CK1

Lactate Dehydrogenase (LDH)

Aminotransferases and liver damage

AminotransferasesAlanine aminotransferase (ALT)Aspartate aminotransferase (AST):

Presence in serum is indicative of liver diseaseOccupational medicine: Liver damage generated by toxic solvent.Monitor liver enzymes when take some lipid-lowering, anti-diabetic and anti-hypertension drugs.


Diagnostic toolsAnalytical toolsTherapeutic tools

Coupled Assays

When the product(s) and substrate (s) of the reaction of interest cannot be easily measured, it is often necessary to couple the reaction to a second reaction. Eg. Reaction of interest

Glucose + ATP Glucose-6-phosphate + ADPglucokinase

Glu-6-P + NADP 6-phosphogluconolactone + NADPH + H+

Glu-6-P dehydrogenase

A340

Glucose Detection

Glucose + H2O + O2 gluconic acid + H2O2

H2O2 + dye H2O + colored dye

Glucose oxidase

Peroxidase

Glucose Detection Strip (Reagents / enzyme immobilized on a strip)

ELISA (Enzyme-Linked ImmunoadsorbentAssay)Eg. Detection of antibodies by ELISA

Medical Microbiology by Elliott, Hastings, Desselberger (Fig. 25-3)

Enzyme

colored products


Diagnostic toolsAnalytical toolsTherapeutic tools

Therapeutic toolsDrugs: enzyme inhibitorsStreptokinase: a plasminogen activator; to clear blood clots by stimulating the conversion of plasminogen to plasmin.

• Acute Myocardial Infraction • Pulmonary Embolism (blood clots block an artery in the lungs)• Thrombosis (blood clots in veins deep inside the legs) Plasminogen

Plasmin

Fibrinogen FibrinFibrin degradation products

Degradation products

+ +

+ streptokinase

Therapeutic tools

Abzyme (Engineered Catalytic Antibodies): tumors are selectively destroyed while healthy cells are spared from the toxic affect of cancer drugs

1. Abs binds the tumor cells with high affinity

2. Prodrug is introduced into the bloodstream, but only becomes activated in the vicinity of the targeted antibody.

LSM1101_Enzyme3

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