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Ch. 19 - Enzymes and Vitamins

• Living systems are shaped by an enormous variety of biochemical reactions nearly all of which are mediated by a series of remarkable biological catalysts known as enzymes.

• Enzymology (study of enzymes) and biochemistry evolved together from the 19th century investigation of fermentation.

• “enzyme” – Greek: en, in + zyme, yeast

• E. Buchner showed that alcohol fermentation (ethanol production from glucose) could be carried out using cell-free yeast extract.

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What are enzymes?• Enzymes are biological catalysts.

• Would you expect enzymes to be fibrous or globular proteins?

• They are extremely effective, increasing reaction rates from 106 to 1012 times.

• Most enzymes act specifically with only one reactant (called a substrate) to produce products

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• Enzymes facillitate chemical reactions in an active site, a pocket in an enzyme with the specific shape and chemical makeup necessary to bind a substrate and where the reaction takes place. The amino acids His, Cys, Asp, Arg, and Glu participate in 65% of all active sites.

•A living cell has a set of some 3,000 enzymes that it is genetically programmed to produce. If even one enzyme is missing or defective, the results can be disastrous.

•Enzymes are used in household products including meat tenderizer, facial treatments, laundry products, and contact lens cleaners.

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2 H2O2 (l) 2 H2O (l) + O2(g)

• An example of an enzyme found in many living organisms is catalase

Each molecule of catalase is a tetramer of four polypeptide chains. Each chain is composed of more than 500 amino acids. Located within this tetramer are four porphyrin heme groups much like the familiar hemoglobins, cytochromes, chlorophylls and nitrogen-fixing enzymes in legumes. The heme group is responsible for catalase’s enzymatic

activity.

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What are some general characteristics of an enzyme?

• Specificity

Enzymes have a great degree of specificity with respect to the identities of the substrate (reactants) and their products than do chemical catalysts.

Absolute specificity: Enzymes catalyzing of the reaction of one

and only one substance

Relative specificity:Enzymes that catalyze the reaction of

several structurally related substances

Stereochemical specificity:Enzymes that are able to distinguish

between stereoisomers

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The specificity of an enzyme for The specificity of an enzyme for one of two enantiomers is a one of two enantiomers is a matter of fit. One enantiomer fits matter of fit. One enantiomer fits better into the active site of the better into the active site of the enzyme than the other enzyme than the other enantiomer. An enzyme catalyzes enantiomer. An enzyme catalyzes reaction of the enantiomer that fits reaction of the enantiomer that fits better into the active site of the better into the active site of the

enzyme.enzyme.

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Muscle Relaxants and Enzyme Specificity

The mode of action of succinylcholine, a muscle relaxant used during minor surgery, utilizes the substrate specificity of the enzyme, acetylcholinesterase.

+ H2OOH

O

O

N+

O acetylcholine-esterase enzyme

+ OH

N+

O

N+

O

N+

O

O

Succinylcholine

Acetylcholine

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• Catalytic Efficiency

Rates of enzymatically catalyzed reactions are typically 106 to 1012 times greater than those of the corresponding uncatalyzed reactions

Turnover number: The number of molecules of substrate acted on by one molecule of enzyme per minute

Ex: Carbonic anhydrase converts carbon dioxide to bicarbonate at a rate of 36 million molecules per minute.

CO2 + H2O HCO3- + H+

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• Milder Reaction Conditions

Reactions occur under relatively mild conditions

Temperature below 100°C, atmospheric pressure, and nearly neutral pH

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• Cofactors

Many enzymes are conjugated proteins that require non-protein portions known as cofactors.

Some cofactors are metal ions.

Carboxypetptidase is an enzyme that requires a Zn 2+ ion as a cofactor. This enzyme hydrolyzes the first amide bond at the C-terminal end of peptides. Carboxypeptidase is synthesized in the pancreas and secreted in the small intestine.

A “space-filled” representation of carboxypeptidase

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A proposed model of the active-site chemistry of carboxypeptidase

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Others cofactors are non-protein organic molecules called coenzymes.

Lactate dehydrogenase is an enzyme that requires the coenzyme NAD+ /NADH for enzymatic function.

Which species have been reduced? Oxidized?

HO

CO2-

CH3

H + NAD+C

CO2-

CH3

O + NADH + H+

Lactatedehydrogenase

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Remember, enzymes catalyze both the forward and the reverse processes.

Biochemical reactions are often represented with the coenzymes/ enzymes written in conjuction with the equation “reacts to form” arrow.

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Structure of NAD+ and NADH

NAD+

Nicotinamide adenine dinucleotideNADHNicotinamide adenine dinucleotide

N

NN

N

NH2

O

HOH

HH

HH

OP-O

O

N

O

HOH

HH

HH

OP-O

O

O

NH2

OH H

N

NN

N

NH2

O

HOH

HH

HH

OP-O

O

N

O

HOH

HH

HH

OP-O

O

O

NH2

OH

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• Capacity for Regulation

• Cells control the rates of reactions and the amount of any given product formed by regulating the action of the enzyme.

Mechanisms for regulatory process:

Allosteric and feedback control

Covalent modification of the enzyme

Variation of enzyme concentration

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How are enzymes classified?

Enzymes may be classified according to the type of reaction that they catalyze.

Six main classes:

1. Oxidoreductase: redox reactions

2. Transferase: transfer functional groups

3. Hydrolase: hydrolysis reactions

4. Lyase: addition and elimination reactions

5. Isomerase: isomerization reaction

6. Ligase: bond formation coupled with ATP

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Oxidoreductases

• Catalyze oxidation-reduction (redox) reactions, most commonly addition or removal of oxygen or hydrogen.

• Requires coenzyme

HO

CO2-

CH3

H + NAD+C

CO2-

CH3

O + NADH + H+

Lactatedehydrogenase

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Transferases• Catalyze transfer of a functional group from one

molecule to another• Kinase applied to enzymes that catalyze transfer of

terminal phosphate group

O

CH2

HH

OH

H

OHH

OH

OH

H

HO

O

CH2

HH

OH

H

OHH

OH

OH

H

OP

O

O-

-O

hexokinase

ATP ADP

Mg2+

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Hydrolases• Catalyze the hydrolysis of substrate – the

breaking of bond with addition of water.

• These reactions are important in the digestive process.

H2C

HC

H2C

O

O

O

C

O

(CH2)nCH3

C

O

(CH2)nCH3

C

O

(CH2)nCH3

+ 3 H2OLipase

H2C

HC

H2C

OH

OH

OH

+

3 CH3(CH2)nCO2H

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Isomerases

• Catalyze the isomerization of a substrate in reactions that have one substrate and one product

• Rearranges of the functional groups within a molecule (catalyst converts one isomer in to the other)

HO

CO2-

H phosphoglyceratemutase

O

H H

P-O

O-

O

HO

CO2-

OH

H H

O-

PO

O-

O

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Lyases• Catalyze the addition of groups such as H2O, CO2, or NH3 to a

double bond or reverse reaction in which a molecule is eliminated to create a double bond

O C O + H OH O

OH

OH

carbonicanhydrase

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Ligases

• Catalyze the bonding of two substrate molecules

• reaction where C-C, C-S, C-O, or C-N bond is made or broken

• Accompanied by ATP-ADP conversion (release of energy drives reaction)

H3C C

O

C

O

O-

CO2

H2C C C

O

O-

+ Pi H++ + ATP+ ADP +

pyruvate carboxylase

O

-O C

O

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Subclasses and Types of Reaction• Oxidoreductase

– Oxidase Oxidation of a substrate

– Reductase Reduction of a substrate

– Dehydrogenase: Introduction of a double bond (C-C or C-O)

• Transferase – Transaminase Transfer amino

groups– Kinase Transfers a phosphate

group• Hydrolyase

– Lipase Hydrolyzes ester groups of lipids

– Protease Hydrolyzes amide bonds of proteins

– Nuclease Hydrolyzes phosphate esters in nucleic acids

• Lyase – Dehydrase Loss of water

from a substrate– Decarboxylase Loss of

carbon dioxide from a substrate

• Ligase – Synthetase Formation of a

new C-C bond from two substrates

– Carboxylase Formation of a new C-C bond with carbon dioxide.

• Isomerase – Epimerase Isomerization

of a chiral carboncenter

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How are enzymes named?

• Common Names– Derived from the name of the substrate

• Urease• Lactase

– Derived from the reactions they catalyze• Dehydrogenase• Decarboxylase

– Historical Names• Have no relationship to either substrate or reaction• catalase, pepsin

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• Systematic Naming

– Unambiguous (often very long)– Specifies:

• Substrate (substance acted on)• Functional group• Type of reaction catalyzed

– Names end in –ase

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Example

• Systematic Name: Urea amidohydrolase– Substrate: urease– Functional group: amide– Type of reaction: hydrolysis

• Common name: Urease

H2N C

O

NH2

+ H2O CO2 + 2 NH3

enzyme

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Can you name the missing enzymes?

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How do enzymes work?

• General Theory– Substrate and enzyme molecules come into contact and interact

over only a small region of the enzyme surface (active site)

– The substrate bonds to active site via temporay non-covalent interactions and covalent interactions (less frequently).

– An enzyme-substrate complex (ES) is formed when a substrate and enzyme bond.

– The substrate is destabilzed in the ES complex by various non-covalent forces.

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• Two models are proposed to represent the interaction between substrates and enzymes.

These are:

– Lock-and-key model: The substrate is described as fitting into the active site as a key fits into a lock.

– Induced-fit-model: The enzyme has a flexible active site that changes shape to accommodate the substrate and facilitate the reaction.

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• Mechanisms of Catalysis

The chemistry at the active site of an enzyme is the most important factor in catalytic effect of an enzyme.

• Proximity Effect – the enzyme positions the reactant(s) for a reaction

• Orientation Effect – positioning of the reactant(s) in the active site allows for optimum orientation

• Catalytic Effect – atoms at the active site provide structural features that facillitate the chemistry of the reaction

• Energy Effect – the activation energy requirements for the reaction are reduced due to any combination of the above

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Hydrolysis of a peptide bond by chymotrypsin

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How is enzyme activity measured?

• Experiments that measure enzyme activity are called assays.

• Assays for blood enzymes are performed in medical laboratories– Some assays determine how fast the characteristic color of a

product forms or the color of a substrate decreases

– Some assays are based on reactions in which protons are produced or used up. This type of enzyme activity can be followed by measuring how fast the pH of the reacting mixture changes with time.

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Diagnostically Useful Assays

• Alanine Transamininase (AST)– Hepatitis

• Lactate dehydrogenase (LDH)– Heart attacks, liver damage

• Acid phosphatase– Prostate cancer

• Creatine Kinase (CK)– Heart attacks

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What are some factors that effect the rate of enzyme catalyzed reactions?

• Enzyme Concentration

• Substrate Concentration

• Temperature

• pH

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Effect of Concentration

• Substrate concentration: At low substrate concentration, the reaction rate is directly proportional to the substrate concentration. With increasing substrate concentration, the rate drops off as more of the active sites are occupied.

•Enzyme concentration: The reaction rate varies directly with the enzyme concentration as long as the substrate concentration does not become a limitation.

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Change of reaction rate with substrate concentration when enzyme Change of reaction rate with substrate concentration when enzyme concentration is constant.concentration is constant.

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Change of reaction rate with enzyme concentration with Change of reaction rate with enzyme concentration with no limit on the substrate concentration.no limit on the substrate concentration.

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Effect of Temperature and pH

• Effect of Temperature: Increase in temperature increases the rate of enzyme catalyzed reactions. The rates reach a maximum and then begins to decrease. The decrease in rate at higher temperature is due to denaturation of enzymes.

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Effect of temperature on reaction rateEffect of temperature on reaction rate

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• Effect of pH: The catalytic activity of enzymes depends on pH and usually has a well defined optimum point for maximum catalytic activity.

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How is enzyme activity regulated?

Any process that starts or increase the activity of an enzyme is activation.

Any process that stops or slows the activity of an enzyme is inhibition.

We will see that there are several modes of enzyme regulation:

• Feedback • Allosterism • Inhibition • Covalent Modification and Genetic Control

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Two of the mechanisms that control the enzymes activity are:

• Feedback control: Regulation of an enzyme’s activity by the product of a reaction later in a pathway.

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Example of Feedback Control: Synthesis of Isoleucine

• Threonine deaminase (enzyme that catalyzes 1st step) is subject to inhibition by the final product (isoleucine)

• Isoleucine binds to a different site on the enzyme and changes the conformation and threonine binds poorly

• As the concentration of isoleucine increases, the enzyme activity drops.

H

C

C

CH3

+H3N COO-

OHH

H

C

C

CH2

+H3N COO-

CH3H

CH3

threoninedeaminase

threonineisoleucine

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• Allosteric control: Activity of an enzyme is controlled by the binding of an activator or inhibitor at a location other than the active site.

Positive allosteric regulator - changes the active site making the enzyme a better catalyst (rate accelerates).

Negative allosteric regulator - changes the active site so that the enzyme becomes a less effective catalyst (rate decreases).

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A positive regulator changes the activity site so that the enzyme becomes a better catalyst and rate accelerates.

A negative regulator changes the activity site so that the enzyme becomes less effective catalyst and rate slows down.

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• Covalent modification Enzyme availability can be controlled by storing the enzyme in its

inactive form called zymogens or proenzymes. When the active enzyme is needed, the stored zymogen is released from storage and activated at the location of the reaction.

Activation of a zymogen requires a covalent modication of its structure.

Examples:– Pepsinogen pepsin

• Digestion of proteins

– Prothrombin thrombin• Blood clotting

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A 3D rendition of the protease pepsin and its zymogen form pepsinogen.

Pepsinogen has 44 extra amino acids (shown in green) which are removed when the enzymatic activity of pepsin is needed.

Pepsinogen

Pepsin

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The inhibition of an enzyme can be reversible or irreversible.

In reversible inhibition, the inhibitor can leave, restoring the enzyme to its uninhibited level of activity.

In irreversible inhibition, the inhibitor remains permanently bound to the enzyme and the enzyme is permanently inhibited.

Inhibitions are further classified as competitive or non-competitive.

Non- competitive inhibition if the inhibitor does not compete with a substrate for the active site.

• Inhibition

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Competitive inhibition - the inhibitor competes with a substrate for the active site.

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Noncompetitive inhibition - the inhibitor binds elsewhere and not to the active site.

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• Genetic control

The supply and synthesis of enzymes is regulated by genes.

Enzymes required at different stages of development are under genetic control.

Mechanisms controlled by hormones can accelerates or decelerates enzyme synthesis.

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What are vitamins?

• Vitamins are organic molecules that function in a wide variety of capacities within the body.

• The most prominent function is as cofactors for enzymatic reactions.

• The distinguishing feature of the vitamins is that they generally cannot be synthesized by mammalian cells and, therefore, must be supplied in the diet.

• Vitamins can be classified as water soluble or fat soluble

• Several vitamins work as antioxidants to protect biomolecules from

damage by free radicals.

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.Water Soluble Vitamins

• Thiamin (B1) • Riboflavin (B2) • Niacin (B3) • Pantothenic Acid (B5) • Pyridoxal,

Pyridoxamine, Pyridoxine (B6)

• Biotin • Cobalamin (B12) • Folic Acid • Ascorbic Acid

Fat Soluble Vitamins

• Vitamin A • Vitamin D • Vitamin E • Vitamin K

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Niacin

• Niacin (nicotinic acid and nicotinamide) is also known as vitamin B3

• Niacin is required for the synthesis of the active forms of vitamin B3, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+).

• Both NAD+ and NADP+ function as cofactors for numerous dehydrogenase enzymes

                          

       

                            

    

Nicotinamide Nicotinic Acid