HL Chemistry - Option B: Human Biochemistry
Enzymes
Part 1
Overview of Enzymes
Enzyme Fundamentals• Enzymes are protein complexes that Enzymes are protein complexes that speed upspeed up biochemical biochemical
reactions by reactions by lowering the activation energylowering the activation energy• Enzymes accelerate reactions by facilitating the formation of the Enzymes accelerate reactions by facilitating the formation of the
transition statetransition state• The position of the equilibrium, enthalpy of reaction, and free The position of the equilibrium, enthalpy of reaction, and free
energy of the reaction are unchanged by an enzymeenergy of the reaction are unchanged by an enzyme• The enzymes themselves are the same after the reaction as they The enzymes themselves are the same after the reaction as they
was beforewas before• Enzymes are powerful and highly specific catalystsEnzymes are powerful and highly specific catalysts• Free energy is a useful thermodynamic function for understanding Free energy is a useful thermodynamic function for understanding
enzymesenzymes• The The Michaelis-MentenMichaelis-Menten model accounts for the kinetic properties of model accounts for the kinetic properties of
many enzymesmany enzymes• Enzymes can be inhibited by specific moleculesEnzymes can be inhibited by specific molecules• Vitamins are often precursors to coenzymesVitamins 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
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
And More Enzyme Terminology• 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
And Yet More Enzyme Terminology• 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
Examples of Enzyme Cofactors • Apoenzyme +Apoenzyme +cofactor =cofactor =holoenzyme holoenzyme
• Cofactors oftenCofactors oftenderived fromderived fromvitamins vitamins
• When tightlyWhen tightlybound to bound to enzyme,enzyme,cofactor =cofactor =prosthetic groupprosthetic group
• Many enzymesMany enzymesuse sameuse samecofactorcofactor
Cofactor Function
and Co-Enzymes!
Enzymes Cofactors may be Metal IonsMetal ions are present in trace amounts (e.g. Mg+2, Ca+2, Zn+2)
Enzyme CofactorsCoenzyme: 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 anypeptide bond
Close Up of Thrombin Cleavage Site
The specificity of an enzyme is due to the precise The specificity of an enzyme is due to the precise interaction of substrate with the enzyme.interaction of substrate with the enzyme. This is a result of This is a result of
the unique three-dimensional structure of the enzyme the unique three-dimensional structure of the enzyme
Enzyme ClassesMost 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 2.7.4.42 = 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
• A substrate must be brought into favorable orientation at a specific region of the enzyme called the active site
Evidence Supporting ES Complex Formation:
1. An enzyme-catalyzed reaction has a maximal velocity suggesting the formation of a discrete ES complex (at high S concentrations catalytic sites are filled)
2. X-ray crystallography has provided high resolution images of substrates and substrate analogs bound to the active sites of many enzymes
3. Spectroscopic characteristics of many enzymes and substrates change on formation of an ES complex
The Active Site of an Enzyme1. The active site is the region that binds the substrates (& cofactors if any)
2. It contains the residues that directly participate in the making & breaking of bonds (these residues are called catalytic groups)
3. The interaction of the enzyme and substrate at the active site promotes the formation of the transition state
4. The active site is the region that most directly lowers the Free Energy (G‡) of the reaction - resulting in rate enhancement of the reaction
Common Features of Active SitesEnzymes differ widely in, structure, specificity, & mode of catalysis, yet, active site have common features:
1. The active site is a 3-dimensional cleft formed by groups that come from different parts of the amino acid sequence
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
3. Active sites are clefts or crevices – they exclude H2O
4. Substrates are bound to enzymes by multiple weak attractions such as electrostatic interactions, hydrogen bonds, Van der Waals forces, & hydrophobic interactions
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 (Vmax).
Vmax
Michaelis-Menten Kinetics
V0 = Vmax x [S]/([S] + Km)
Michaelis – Menten EquationMichaelis – Menten Equation
V0 = moles of product formed per sec. when [P] is low (close to zero time); V0 varies with [S]
E + S ES E + P Michaelis-Menten Model
Km = [S] when V0 = Vmax/2 Km 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 ConditionsSteady-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 zerotime), thus, use V0 for initial reaction velocity
At pre-steady state, we ignore the back reactions
Michaelis-Menten KineticsEnzyme 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
ES complex is a necessary intermediate!
k2
Start with: V0 = k3[ES], and derive, V0 = Vmax x[S]/([S] + Km)
This equation accounts for graphical data
At low [S]: ([S] < Km), V0 = (Vmax/Km)[S]At high [S]: ([S] > Km), V0 = Vmax
When [S] = Km: V0 = Vmax/2
Thus, Km = substrate concentration at which the reaction rate (V0) is half max
k1 k3
Range of Km values
Km provides approximation of [S] in vivo for many enzymes
Lineweaver-Burk plot (double-reciprocal)
• Due to the asymptotic approach to Vmax 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-BurkLineweaver-Burk relationship:
• 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 = Vmax x [S]/([S] + Km)Michaelis – Menten Equation
1/V = (Km /Vmax x 1/[S]) + 1/ Vmax )
Lineweaver-Burk Plot (double-reciprocal)
1/V = (Km /Vmax x 1/[S]) + 1/ Vmax )
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 overcomeinhibition
• Vmax is then attainable, and Km is increased
← Ki = dissociation constant for inhibitor
Competitive Inhibitor Lineweaver-Burk Plot
Vmax is unaltered, but Km is increased!
Kinetics of Non-Competitive Inhibition
Unlike competitive inhibition, increasing [S] can notovercome inhibition in the non-competitive case
Non- Competitive Inhibitor Lineweaver-Burk Plot
Km is unaltered, but Vmax 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)Ascorbic acid is a reducing agent (an antioxidant)
• B series vitamins are components of coenzymes,B series vitamins are components of coenzymes,• They must be modified before they can serve their They must be modified before they can serve their functionsfunctions
A few facts: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
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
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.