HL Chemistry - Option B: Human Biochemistry

Enzymes

Part 1

Overview of Enzymes

Enzyme Fundamentals

• Enzymes are protein complexes that speed up biochemical reactions by lowering the activation energy
• Enzymes accelerate reactions by facilitating the formation of the transition state
• The position of the equilibrium, enthalpy of reaction, and free energy of the reaction are unchanged by an enzyme
• The enzymes themselves are the same after the reaction as they was before
• Enzymes are powerful and highly specific catalysts
• Free energy is a useful thermodynamic function for understanding enzymes
• The Michaelis-Menten model accounts for the kinetic properties of many enzymes
• Enzymes can be inhibited by specific molecules
• Vitamins are often precursors to coenzymes

Some Enzyme Terminology

• Enzyme – a biomolecule that catalyzes biochemical reaction by lowering activation energy
• Substrate – the substance that undergoes a chemical change by an enzyme
• Absolute Specificity – the characteristic that an enzyme acts on only one substrate
• Relative Specificity – the characteristic that an enzyme acts on several structurally related substrates
• Stereochemical Specificity – an enzyme's ability to distinguish between stereoisomers
• Cofactor – a nonprotein molecule or ion required by an enzyme for catalytic activity
• Coenzyme – an organic molecule required by an enzyme for catalytic activity

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More Enzyme Terminology

• Apoenzyme – a catalytically inactive protein formed by removal of the cofactor from an active enzyme
• Active Site – the location on an enzyme where a substrate is bound and catalysis occurs
• Enzyme Activity – the rate at which an enzyme catalyzes a reaction
• Turnover Number – the number of molecules of substrate acted upon by one molecule of enzyme per minute
• Enzyme International Unit (IU) – a quantity of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under specified conditions
• Optimum Temperature – the temperature at which enzyme activity is highest

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And More Enzyme Terminology

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• Optimum pH - the pH at which enzyme activity is highest
• Extremozyme – an enzyme that thrive in extreme environments
• Enzyme Inhibitor – a substance that decreases the activity of an enzyme
• Competitive Inhibitor – an inhibitor that binds to the active site of an enzyme
• Noncompetitive Inhibitor – an inhibitor that binds at a location other than the enzyme’s active site
• Zymogen (proenzyme) – the inactive enzyme precursor
• Modulator – a substance that binds to an enzyme at a location other than the active site that alters the enzyme's catalytic activity

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And Yet More Enzyme Terminology

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• Allosteric Enzyme – an enzyme with a quaternary structure whose activity is changes by the binding of a modulator
• Activator – a substance that binds to the allosteric enzyme and increases its activity
• Feedback Inhibition – a process in which the end product of a sequence of enzyme catalyzed reaction inhibits an earlier step in the process
• Enzyme Induction – the synthesis of enzyme in response to a cellular need
• Isoenzyme – a slightly different form of the same enzyme produced by different tissues
• Holoenzyme – apoenzyme + cofactor

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Examples of Enzyme Cofactors

• Apoenzyme +

cofactor =

holoenzyme

• Cofactors often

derived from

vitamins

• When tightly

bound to enzyme,

cofactor =

prosthetic group

• Many enzymes

use same

cofactor

Cofactor Function

and Co-Enzymes!

Enzymes Cofactors may be Metal Ions

Metal ions are present in trace amounts (e.g. Mg⁺², Ca⁺², Zn⁺²)

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Enzyme Cofactors

Coenzyme: a non-protein organic (may be a vitamin)

Example of Enzymatic Catalysis: Hydration of CO₂

• This reaction is catalyzed by carbonic anhydrase (10⁶ molecules of CO₂ per sec: 10⁷ times faster than without enzyme!)
• Speeds up transfer of CO₂ from tissue to blood to alveolar air

Substrates

Product

No wasteful by-products!

Selected Enzyme Reaction Rates

Example of Enzyme Substrate Specificity: proteolysis

• Enzymatic hydrolysis of a specific peptide bond in vivo

Substrates

Products

Example of Enzyme Substrate Specificity: (continued)

• Example (A): Trypsin cleavage site at Lys or Arg (digestive enzyme)

• Example (B): Thrombin cleavage site at Arg only

(blood clotting enzyme)

• One particular enzyme, Subtilisin, will cleave any

peptide bond

Close Up of Thrombin Cleavage Site

The specificity of an enzyme is due to the precise interaction of substrate with the enzyme. This is a result of the unique three-dimensional structure of the enzyme

Enzyme Classes

Most named for substrates & for reactions, with suffix “ase”

(e.g.: ATPase breaks down ATP, ATP synthase makes ATP)

• 1964, classification & nomenclature of enzymes was developed by the International Enzyme Commission (IEC):

e.g. Nucleoside Monophosphate (NMP) Kinase = IEC 2.7.4.4

2 = class, 7 = phosphoryl group, 4 = phosphate acceptor,

4 = precise acceptor (NMP)

Part 2

Enzyme Kinetics

The Enzyme-Substrate Complex

• The catalytic power of enzymes is derived from the formation of the transition states in enzyme-substrate (ES) complexes

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• A substrate must be brought into favorable orientation at a specific region of the enzyme called the active site

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Evidence Supporting ES Complex Formation:

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• An enzyme-catalyzed reaction has a maximal velocity suggesting the formation of a discrete ES complex (at high S concentrations catalytic sites are filled)

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2. X-ray crystallography has provided high resolution images of substrates and substrate analogs bound to the active sites of many enzymes

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3. Spectroscopic characteristics of many enzymes and substrates change on formation of an ES complex

The Active Site of an Enzyme

• The active site is the region that binds the substrates (&

cofactors if any)

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2. It contains the residues that directly participate in the making &

breaking of bonds (these residues are called catalytic groups)

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• The interaction of the enzyme and substrate at the active site

promotes the formation of the transition state

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4. The active site is the region that most directly lowers the Free Energy (ΔG^‡) of the reaction - resulting in rate enhancement of the reaction

Common Features of Active Sites

Enzymes differ widely in, structure, specificity, & mode of catalysis, yet, active site have common features:

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• The active site is a 3-dimensional cleft formed by groups that come from different parts of the amino acid sequence

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2. The active site takes up a relatively small part of the total volume of an enzyme. Why are enzymes so big? Answer: Scaffolding, regulatory sites, interaction sites for other proteins, & channels

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3. Active sites are clefts or crevices – they exclude H₂O

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4. Substrates are bound to enzymes by multiple weak attractions such as electrostatic interactions, hydrogen bonds, Van der Waals forces, & hydrophobic interactions

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5. The specificity of binding depends on the precisely defined arrangement of atoms at the active site

Active Sites are Composed of Distant Residues

The Enzyme – Substrate Complex Is Usually Stabilized by Hydrogen-Bonds

EXAMPLE:

Ribonuclease

(cleaves RNA)

Lock-and-Key (ES) Model

This model assumes that a unique substrate binds to the active site. Thus, there must be a 1:1 ratio between substrates and enzymes. This is in fact not true, since there are many more substrates than enzymes. Therefore this model is not currently favored by most biochemists.

Induced Fit (ES) Model

In this model, the active site can change shape slightly to accommodate substrates with similar shapes and charges. This model is favored by most biochemists.

Comparison of Lock & Key vs. Induced Fit Models

Diagrammatic representation of the two (ES) binding theories illustrates how the Lock & Key Theory (a) yields a 1:1 ratio of substrate to enzyme, whereas the Induced Fit Model (b) suggests the enzyme can accommodate several types of substrates.

Enzyme - Catalyzed Reactions: maximal velocity

Under initial conditions the plot is linear and first order (or pseudo-first order). After the product concentration starts to build up, the reverse reaction becomes more important and the reaction velocity asymptotically approaches the maximal velocity (V_max).

Rate = k[enzyme]¹

(when [enzyme] << [substrate])

V_max

Michaelis-Menten Kinetics

V₀ = V_max x [S]/([S] + K_m)

Michaelis – Menten Equation

V₀ = moles of product formed per sec. when [P] is low (close to zero time); V₀ varies with [S]

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E + S ⇔ ES ⇒ E + P

Michaelis-Menten Model

K_m = [S] when V₀ = V_max/2

K_m is the “Michaelis Constant”

It is a function of the kinetic rate constants

Initial velocity V₀ (when [P] is low)

(Ignore the back reaction!)

Steady-State & Pre-Steady-State Conditions

At equilibrium, there is no net change of [S] & [P] or [ES] & [E]

At pre-steady-state,

[P] is low (close to zero

time), thus, use V₀ for

initial reaction velocity

At pre-steady state, we ignore the back reactions

Michaelis-Menten Kinetics

Enzyme kinetics based on the Michaelis-Menten Graph:

At a fixed concentration of enzyme, V₀ is almost linearly proportional to [S] when [S] is small, but is nearly independent of [S] when [S] is large.

Proposed Model: E + S ⇔ ES ⇒ E + P

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ES complex is a necessary intermediate!

k₂

Start with: V₀ = k₃[ES], and derive, V₀ = V_max x[S]/([S] + K_m)

This equation accounts for graphical data

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At low [S]: ([S] < K_m), V₀ = (V_max/K_m)[S]

At high [S]: ([S] > K_m), V₀ = V_max

When [S] = K_m: V₀ = V_max/2

Thus, K_m = substrate concentration at which the reaction rate (V₀) is half max

k₁

k₃

Range of K_m values

K_m provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

• Due to the asymptotic approach to V_max given by Michaelis-Menten Kinetics, it is sometimes very difficult to find the various components in the aforementioned equation
• Rearrangement of the Michaelis-Menten equation gives the Lineweaver-Burk relationship:

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• This is of the form y = mx + b, so a plot of 1/[S] vs.1/V₀ produces a straight line with values as shown on the next slide

V₀ = V_max x [S]/([S] + K_m)

Michaelis – Menten Equation

1/V = (K_m /V_max x 1/[S]) + 1/ V_max )

Lineweaver-Burk Plot (double-reciprocal)

1/V = (K_m /V_max x 1/[S]) + 1/ V_max )

Allosteric Modulation

Allosteric Enzyme Kinetics

• Sigmoidal dependence of V₀ on [S], means the enzyme kinetics are not Michaelis-Menten!

• Enzymes can have multiple subunits

and multiple active sites

• Substrate binding may be cooperative!

Enzyme Inhibition – Competitive vs. Noncompetitive

Kinetics of Competitive Inhibition

• Increase [S] to overcome

inhibition

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• V_max is then attainable, and K_m is increased

← K_i = dissociation constant for inhibitor

Competitive Inhibitor Lineweaver-Burk Plot

V_max is unaltered, but K_m is increased!

Kinetics of Non-Competitive Inhibition

Unlike competitive inhibition, increasing [S] can not

overcome inhibition in the non-competitive case

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Non- Competitive Inhibitor Lineweaver-Burk Plot

K_m is unaltered, but V_max is decreased!

Part 3

A Few Enzyme Applications

Vitamins as Enzymes

Vitamins can either be water soluble or fat soluble

• They play important roles in metabolism
• If too many or too few vitamins are present, disease will result

Vitamins: Water-Soluble

Vitamins: Fat-Soluble

Structures of Some Water-Soluble Vitamins

• Ascorbic acid is a reducing agent (an antioxidant)

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• B series vitamins are components of coenzymes,
• They must be modified before they can serve their functions

A few facts:

Structure of Some Fat-Soluble Vitamins

Enzyme Denaturation

• Enzymes are only functional if they have the proper 3-D structure
• Changes in temperature, pH, salt concentration, metal ion content, and solvent polarity can cause the enzyme to change conformation, and thus become inactive

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Denaturation of an Enzyme with pH or Temperature

Enzymes – Effect of pH on Activity

Enzymes – Effect of Temperature on Activity

Other Enzyme Denaturants

• Temperature & pH are the main sources of enzyme deactivation, but there are other mechanisms as well
• Since proteins have a hydrophobic interior and hydrophilic exterior in aqueous environments, they can be turned inside out if the polarity of the solvent is changed
• Chaotropes such as SDS (sodium dodecyl sulfate), alcohols, urea, guanidine-HCl, and salts change the polarity of the solvent and denature enzymes
• Heavy metals such as mercury, cadmium, nickel, etc. bind to enzymes anywhere they can find an unsaturated nitrogen atom and cause the enzyme to change conformation and become inactive

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Enzymes in Biotechnology

• Biotechnology is defined as the application and harnessing of microorganisms or biological process to produce desired substances.
• Harnessing yeast to aid in fermentation in one of the oldest examples of biotechnology.
• Much of the current research in biotechnology involves genetic engineering.
• Genetic engineering involves removing a gene from one organism and then combining it with the nucleic acid of another to produce a desired chemical product in large quantities.
• Transfer of the human insulin gene to bacteria (E. coli) is a prime example of genetic engineering

More Biotechnology Examples

• Biological detergents have been prepared by splicing the gene for lipolase into aspergillus. The advantage is these detergents save energy (lower washing temperatures), are biodegradable, and pose little risk to the environment.
• Similar work has produced a new enzyme that breaks he glucose chains in cellulose only when a strand of cellulose is mechanically broken. The cleansing action makes fabric appear brand new.
• Large scale production of the natural anti-viral agent interferon has been cloned into yeast.
• Hepatitis B vaccine is prepared by cloning, and work on AIDS and malaria are following a similar trend.

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