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LSM1101 LECTURE 3 Enzyme Regulation/ Application

Dr Deng Lih Wen Dept of Biochemistry

Michaelis-Menton Plot
E+S k1 k-1 ES k2 E+P
Equilibrium assumption Steady state assumption

Vmax vo
[S] < KM

V max [S] V0 = KM + [S]


[S] = KM [S] >> KM

Vmax/2

V max [S] V0 KM

V 0 = V max

Km

[S]
First order

V 0 = V max / 2

Zero order

Double-Reciprocal Plot (Lineweaver-Burk Plot)


V0 = V max [S] KM + [S]

1 KM + [S] = V 0 V max [S]

Competitive Changes Km

1 KM 1 1 = + V 0 V max [S] V max

Y = aX + b

Uncompetitive Both Km and Vmax decrease

Mixed Changes Vmax, may not affect Km

Regulation of Overal Enzyme Activity


Control of enzyme synthesis amounts Control of enzyme activity
Proteolytic cleavage Compartmentation

DNA

transcription

mRNA
turnover

translation

Enzyme

turnover

Amino acid

nucleotide

Allosteric regulation

Covalent modification

Phosphorylation of Glucose

Hexokinase: 4 isoenzymes Hexokinase I, II, III: most of tissues except liver, broad substrate specificity Hexokinase IV (also called Glucokinase): predominate in liver
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 liver Km for glucose ~0.1mM Allow for utilization of glucose even when blood glucose is low (such as brain) Glucokinase (Hexokinase IV) Liver Km for glucose ~10mM Buffering effect on blood glucose

Glucokinase in Hepatocyte
[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.
Fig 8.14, Lippincotts illustrated Reviews in Biochemistry

Hepatocyte (liver cell)

Ways to regulate glucokinase activity

Kinetics property (high Km) Localization (GK-GKRP in nucleus) Transcriptional regulation by insulin
* Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days)

blood glucose level rise Increase insulin release (-cells of pancreas)

About half of the newly secreted insulin is extracted by the liver Insulin promotes transcription of the glucokinase gene, resulting in an increase in liver glucokinase amounts

Allosteric Enzymes
Allosteric 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 activity Negative effectors inhibition Positive effectors activation Homotropic effectors substrate serves as a modulator Heterotropic effectors modulator is different from the substrate

Allosteric Enzymes Undergo Conformational Changes in Response to Modulator Binding

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

Principles of Biochemistry 4th Edition, Fig 6-26

- modulator

+ modulator

Feedback Inhibition
In many pathways, a regulated step is catalyzed by an allosteric enzyme Building 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 inhibition Product 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 sigmoid
curve 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.

positive effector

negative effector

(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.)

K0.5

Feedback inhibition of ATCase regulates Pyrimidine Synthesis


Binds cooperatively to ATCase

inhibition
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 purine and pyrimidine nucleotides, which are required in roughly equal amounts in nucleic acid synthesis.

Regulation of Enzyme Activity

Proteolytic cleavage
transcription translation

Compartmentation

DNA

mRNA
turnover

Enzyme

turnover

Amino acid

nucleotide

Allosteric regulation

Covalent modification

Zymogens (Proenzymes)
Inactive enzyme precursor; cleavage to be activated Examples: Hormones Proteolytic enzymes of the digestive tract Blood clotting

The mature hormone insulin consists of the disulfidelinked A and B chains

Pancreatic and Gastric Zymogens

Origin Stomach Pancreas Pancreas Pancreas Pancreas

Zymogen Pepsinogen Proelastase Trypsinogen Chymotrypsinogen

Active Protease Pepsin Elastase Trypsin Chymotrypsin

Procarboxypeptidase Carboxypeptidase

Fig 19.4, Lippincotts 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


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

The intrinsic and extrinsic pathways converge at Factor Xa. Activation of thrombin promotes the conversion of fibrinogen into fibrin aggregates into ordered filamentous form the clot.
Biochemistry 3rd Ed, Figure 15.4

Regulation of Enzyme Activity

Proteolytic cleavage

Compartmentation

DNA

transcription

mRNA
turnover

translation

Enzyme

turnover

Amino acid

nucleotide

Allosteric regulation

Covalent modification

Covalent Modification

Phosphorylation/Dephosphorylation

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.
Principles of Biochemistry

Regulation of glycogen phosphorylase activity by phosphorylation


(Glucose)n + Pi Glycogen
Glycogen phosphorylase

(glucose)n-1 + glucose 1-phosphate


phosphoglucomutase

glucose 6-phosphate
liver Convert to glucose muscle Glycolysis

Export to other tissues

ATP synthesis

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

Glycogen Phosphorylase
(Allosteric
Regulation and Covalent Modification)
Glycogen phosphorylase

(Glucose)n + Pi

(glucose)n-1 + glucose 1-phosphate


ATP and G6P: negative heterotropic effectors AMP: a positive heterotrophic effector

Covalent control

P inactive Allosteric control AMP G-6-P, ATP active

active

Biochemistry 3rd Ed, Figure 15.15

Industrial Application
Food industry fermented alcohol drinks, lactose free milk, animal feed Cleaning compounds Laundry detergents, color brightening and softening Production of Antibiotics from microorganisms

Industrial Enzymes for Food Industry


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

Lactose free milk


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.

Animal Feed

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

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
Dr Alexander Fleming

"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.

Clinical Applications
Diagnostic tools Analytical tools Therapeutic tools

Measuring Plasma Enzyme Activity


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.

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 = MM
Skeletal muscle: 98% CK3 and 2% CK2 Cardiac muscle: 70% CK3 and 30% CK2 Other tissues: Mainly CK1

Lactate Dehydrogenase (LDH)

Aminotransferases and liver damage


Aminotransferases Alanine aminotransferase (ALT) Aspartate aminotransferase (AST): Presence in serum is indicative of liver disease Occupational medicine: Liver damage generated by toxic solvent. Monitor liver enzymes when take some lipid-lowering, anti-diabetic and antihypertension drugs.

Clinical Applications
Diagnostic tools Analytical tools Therapeutic 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
glucokinase
Glu-6-P dehydrogenase

Glucose-6-phosphate + ADP

Glu-6-P + NADP

6-phosphogluconolactone + NADPH + H+

A340

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

Glucose + H2O + O2 H2O2 + dye

Glucose oxidase

gluconic acid + H2O2

Peroxidase

H2O + colored dye

ELISA (Enzyme-Linked Immunoadsorbent Assay)


Eg. Detection of antibodies by ELISA

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

colored products Enzyme

Clinical Applications
Diagnostic tools Analytical tools Therapeutic tools

Therapeutic tools
Drugs: enzyme inhibitors Streptokinase: 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 streptokinase

+
Plasmin

+
Degradation products Fibrinogen

+
Fibrin Fibrin degradation products

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.