BTE 417 Enzyme Biotechnology

BTE 417 Enzyme Biotechnology
Enzymes

Part 1 Overview of Enzymes

The study history of enzymes
Enzymes: Biological catalysts that promote and speed up chemical reactions without themselves being altered (consumed) in the process. They determine the patterns of transformations for chemicals, as well as forms of energy in the living organisms.

The discovery of enzymes as the biocatalysts (1)
Both enzymology and biochemistry were evolved from the 19th century investigation on the nature of animal digestion and fermentation. Biochemical reactions could not be reproduced in the lab initially and was thought (e.g., Louis Pasteur) to occur by the action of a “vital force”. The idea of “catalytic force” or “contact substance” promoting fermentation was introduced in about 1830s. Addition of alcohol to an aqueous extract of malt (geminating barley) and saliva precipitated a flocculent material which liquefied starch paste and converted it into sugar, this material was named diastase (1833) (later amylase).

Pepsin was discovered as the active principle in the acid extract of gastric mucosa causing the dissolution of coagulated egg white (1834). Other “soluble ferments” discovered in the 19th century include trypsin (1857), invertin (later invertase and sucrase, 1864), papain (“vegetable trypsin”, 1879), etc. The term Enzyme (something in yeast) ” was first coined for such “unorganized ferments by Kühne in 1876. Enzymes for alcoholic fermentation were found to be active in cell free extracts of yeast (1897, Eduard Buchner): fermentation is a chemical process, not a vital process.

Relationship of initial velocity (V0) and substrate concentration (S) was examined. A mathematical description was established for the kinetics of enzyme action (Michaelis and Menten, 1913). Weak-bonding interactions between the enzymes and their substrates were proposed to distort the substrate and catalyze a reaction (Haldane, 1930s). Before it was known that enzymes are proteins!!! John Burdon Sanderson Haldane ( ) Leonor Michaelis ( ) Maud Menten ( ) A British Geneticist A German A Canadian

The Nobel Prize in Chemistry 1907 Eduard Buchner Germany Landwirtschaftliche Hochschule (Agricultural College) Berlin, Germany b. 1860, d "for his biochemical researches and his discovery of cell-free fermentation"

Enzyme specificity was revealed by studying sugar conversion (Emil Fischer, 1890S)
Sugars of known structure were synthesized and used as substrates of enzymes. The a-methylglucoside was found to be hydrolyzed by invertin, but not by emulsin, whereas the b-methylglucoside was cleaved by emulsin, but not by invertin: the enzyme and the glucoside was considered to fit (complement) each other like a lock and a key. Formation of an ES complex was proposed (1894).

Enzymes were found to be proteins
The question of homogeneity of the enzyme preparations frustrated the field of enzymology for many decades. Nitrogen content analysis and various color tests (for proteins) led to contradictory results. Filterable coenzymes (co-ferments) were discovered in Buchner’s zymase (Harden and Young, 1906). Enzymes were thought to be small reactive molecules adsorbed on inactive colloidal material, including proteins ( as by R. Willstätter in the 1920s). Urease (1926, Sumner) and pepsin (1930, Northrop) were crystallized and found to be solely made of proteins.

Urease crystals ( X 728) Sumner, J. B
Urease crystals ( X 728) Sumner, J. B. (1926) “ The isolation and crystallization of the enzyme urease” J. Biol. Chem. 69:

Pepsin crystals (X90) Northrop, J. H. (1930) “Crystallin pepsin, 1:
Isolation and tests of purity” J. Gen . Physiol. 13:

The Nobel Prize in Chemistry 1946
The Nobel Prize in Chemistry 1946 “ for his discovery that enzymes can be crystallized" "for their preparation of enzymes and virus proteins in a pure form" James Batcheller Sumner John Howard Northrop Wendell Meredith Stanley 1/2 of the prize 1/4 of the prize Cornell University Ithaca, NY, USA Rockefeller Institute for Medical Research Princeton, NJ, USA

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

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 + cofactor = holoenzyme Cofactors often derived from vitamins Many enzymes use same cofactor When tightly bound to enzyme, cofactor = prosthetic group

(Vitamins)

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)

Coenzyme: a non-protein organic (may be a vitamin)
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 No wasteful by-products! Product Substrates

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

Many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to a word/phrase describing their activity Urease (hydrolysis of urea). Transaminase (transfer amino group from one molecule to another). RNA polymerase (formation of RNA by polymerization). But many enzymes are named before this rule was established (e.g., pepsin, trypsin).

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 = class, 7 = phosphoryl group, 4 = phosphate acceptor, 4 = precise acceptor (NMP)

Transfer electrons (hydride ions or H atoms);
Lactate dehydrogenase Transfer electrons (hydride ions or H atoms); play a major role in energy metabolism. e.g., the transfer of a phosphoryl group from ATP to many different acceptors. NMP kinase Chymotrypsin the transfer of functional groups to water. These are direct bond breaking reactions without being attacked by another reactant such as H2O. Fumarase Triose phosphate isomerase In chemical terms, they would be described as elimination and addition reactions. Leading to the formation of C-C, C-S, C-O, C-N bonds. Aminoacyl-tRNA synthetase

Each enzyme is given a systematic name and a unique 4-digit identification number for identification by the Enzyme Commission (E.C.) of IUBMB (since 1964) lactate + NAD pyruvate + NADH + H+ Lactate dehydrogenase (lactate:NAD+ oxidoreductase) 1 Indicates type of cofactor Indicates type of substrate

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: 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 Enzyme
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) 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 Sites
Enzymes differ widely in, structure, specificity, & mode of catalysis, yet, active site have common features: 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 Rate = k[enzyme]1 (when [enzyme] << [substrate])

The effect on V0 of varying [S] is measured when the enzyme
Vmax is extrapolated from the plot: V0 approaches but never quite reaches Vmax. The effect on V0 of varying [S] is measured when the enzyme concentration is held constant. Hyperbolic relationship between V0 and [S] (similar to the O2 binding curve of myoglobin)

A mathematical relationship between V0 and [S] was established ( Michaelis and Menten, 1913; Briggs and Haldane, 1925) k 1 k 2 E + S ES E + P Formation of ES is fast and reversible. The reverse reaction from PS (k-2 step) was assumed to be negligible. The breakdown of ES to product and free enzyme is the rate limiting step for the overall reaction. ES was assumed to be at a steady state: its concentration remains constant over time. Thus V0 = k2[ES] ( )

E + S ES E + P Km is called the Michaelis constant.
Steady-state assumption: Rate of ES formation=rate of ES breakdown k1([Et]-[ES])[S]=k-1[ES] + k2[ES] ([Et] is the total enzyme concentration.) Solve the equation for [ES]: V0 = k2[ES]

The maximum velocity is achieved when all the
enzyme is saturated by substrate, i.e., when [ES] =[Et]. Thus Vmax =k2[Et] The Michaelis-Menten Equation

The Michaelis-Menten Equation nicely
When [S] >> Km When [S] << Km The Michaelis-Menten Equation nicely describes the experimental observations. The substrate concentration at which V0 is half maximal is Km

The Vmax and Km values of a certain enzyme can be measured by the double reciprocal plot (i.e., the Lineweaver-Burk plot).

Michaelis-Menten Kinetics
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 V0 = Vmax x [S]/([S] + Km) Michaelis – Menten Equation 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 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. k1 k3 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

Km provides approximation of [S] in vivo for many enzymes
Range of Km values Km provides approximation of [S] in vivo for many enzymes

The Vmax and Km values of a certain enzyme can be measured by the double reciprocal plot (i.e., the Lineweaver-Burk plot).

Lineweaver-Burk plot (double-reciprocal)
V0 = Vmax x [S]/([S] + Km) Michaelis – Menten Equation 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-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 1/V = (Km /Vmax x 1/[S]) + 1/ Vmax )

The double reciprocal plot: 1/V0 vs 1/[S]

Lineweaver-Burk Plot (double-reciprocal)
1/V = (Km /Vmax x 1/[S]) + 1/ Vmax )

The Michaelis-Menten equation, but not their approximated mechanism applies to a great many enzymes
Most enzymes (except the regulatory enzymes) have been found to follow the Michaelis-Menten kinetics, but their actual mechanisms are usually more complicated (by having more intermediate steps) than the one assumed by Michaelis and menten. The values of Vmax and Km alone provide little information about the number, rates, or chemical nature of discrete steps in the reaction.

The actual meaning of Km depends on the reaction mechanism
For If k2 is rate-limiting, k2<<k-1, Km = k-1/k 1 Km equals to the dissociation constant (Kd) of the ES complex; Km represent a measure of affinity of the enzyme for its substrate in the ES complex. If k2>>k-1, then Km =k2/k1. If k2 and k-1 are comparable, Km is a complex function of all three rate constants. k -1

Vmax is determined by kcat, the rate constant of the rate-limiting step
Vmax = kcat[Et] kcat equals to k2 or k3 or a complex function of both, depending on which is the rate-limiting step. kcat is also called the turnover number: the number of substrate molecules converted to product in a given unit of time per enzyme molecule when the enzyme is saturated with substrate.

40,000,000 molecules of H2O2 are converted
to H2O and O2 by one catalase molecule within one second!

The kinetic parameters kcat and Km are often studied and compared for different enzymes
Km often reflects the normal substrate concentration present in vivo for a certain enzyme. The catalytic efficiency of different enzymes is often compared by comparing their kcat/Km ratios (the specificity constant). when [S]<<Km kcat/Km is an apparent second-order rate constant (with units of M-1S-1), relating the reaction rate to the concentrations of free enzyme and substrate.

The value of kcat/Km has an upper limit (for the perfected enzymes)
It can be no greater than k1. The decomposition of ES to E + P can occur no more frequently that E and S come together to form ES. The most efficient enzymes have kcat/Km values near the diffusion-controlled limit of 108 to 109 M-1S-1.

Catalytic perfection (rate of reaction being
diffusion-controlled) can be achieved by a combination of different values of kcat and Km.

Enzyme-catalyzed reactions of two or more substrates can also be analyzed by the Michaelis-Menten approach Each substrate will have one characteristic Km value. Noncovalent ternary complex (with two substrates bound to the enzyme concurrently) may or may not be formed for the bisubstrate reactions depending on the mechanism. Steady-state kinetics can often help distinguish these two mechanisms.

In those enzyme-catalyzed bisubstrate reactions where a
ternary complex is formed, the two substrates may either bind in a random sequence or in a specific order.

For those reactions where ternary complex is formed:
Maintaining the concentration of one substrate (S2) constant, the double reciprocal plots made by varying the concentration of the other substrate (S1) will intersect.

No ternary complex is formed in the Ping-Pong (or double displacement) mechanism: The first substrate is converted to a product that leaves the enzyme active site before the second substrate enters.

As [S2] increases, Vmax increases,
For enzymes having Ping-Pong mechanisms (ternary complex not formed). Maintaining the concentration of one substrate (S2) constant, the double reciprocal plots made by varying the concentration of the other substrate (S1) will not intersect. As [S2] increases, Vmax increases, as does the Km for S1. S1

Rates of individual steps for an enzyme-catalyzed reaction may be obtained by pre-steady state kinetics The enzyme (of large amount) is used in substrate quantities and the events on the enzyme are directly observed. Rates of many reaction steps may be measured independently. Very rapid mixing and sampling techniques are required (the enzyme and substrate have to be brought together in milliseconds and measurements also be made within short period of time).

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 catalysis can be slowed or halted by specific inhibitors
Enzyme catalysis can be slowed or halted by specific inhibitors. Such inhibitors are important pharmaceutical agents and useful in understanding the action mechanism of enzymes.

Enzyme Inhibition – Competitive vs. Noncompetitive

How would the Km and Vmax be affected?
Competitive inhibitors have structures similar to the substrates and thus inhibit enzyme catalysis by binding to the active site in a reversible way E + P ES E KI EI How would the Km and Vmax be affected? X

The presence of competitive inhibitors alters the Km but not the Vmax of enzymes
Apparent Vmax and Km values: Vmax unchanged, Km increases

Uncompetitive inhibitors binds at a site distinct from the substrate binding site: the inhibitor binds only to the ES complex How would Km and Vmax be affected? E + P E ES Uncompetitive inhibitors are present only for enzymes catalyzing reactions of two or more substrates (with ordered substrate binding): analogs of S2 will act as uncompetitive inhibitor for the enzyme (relative to S1) ESI X

(but Vmax/Km unchanged).
The presence of uncompetitive inhibitors alter both the Km and the Vmax of an enzyme Both Vmax and Km decreases (but Vmax/Km unchanged).

A mixed inhibitor also binds at a site distinct from the substrate binding site, but binds to either E or ES Noncompetitive inhibitor: binding of I does not affect binding of S; Vmax decreases, Km unchanged. E + P E ES EI ESI Mixed inhibitors are present for enzymes of random ordered substrate binding. X

The presence of mixed inhibitors alter both the Km and the Vmax of an enzyme
Vmax decreases, Km increases.

Kinetics of Competitive Inhibition
Increase [S] to overcome inhibition 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 not overcome inhibition in the non-competitive case

Non- Competitive Inhibitor Lineweaver-Burk Plot
Km is unaltered, but Vmax is decreased!

A Few Enzyme Applications
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
A few facts: B series vitamins are components of coenzymes, They must be modified before they can serve their functions Ascorbic acid is a reducing agent (an antioxidant)

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

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