ERT 317 Biochemical Engineering Sem 1, 2015/2016 CHAPTER 1: ENZYME KINETICS AND APPLICATIONS (Part I : Kinetics of Enzyme Catalyzed Reactions) ERT 317 Biochemical Engineering Sem 1, 2015/2016

Role of Bioprocess Engineering exploit advances in biology to create new products design biochemical processes & operate plants develop energy resources Develop new, environmentally friendly, and safer processes to make the biochemical products that people depend on. Work in research and development laboratories, creating polymeric materials with improved performance and durability. Work in manufacturing, making vaccines and antibiotics. Invent new ways to keep our food and water supplies safe.

Bioprocess Engineer’s Task Minimize production of unwanted byproducts Separate the good (product) from the bad (byproducts) Recover the unused reactants Maximize profit, minimize energy consumption Minimize impact on the environment

OPPORTUNITIES FOR BIOPROCESS ENGINEERS pharmaceuticals polymers energy food consumer products biotechnology electronic and optical materials.

OUTLINE Introduction Enzyme Structure Enzyme Function Enzyme Kinetics A)Michaelis –Menten Kinetics B) The Rapid Equilibrium Assumption C) The Quasi-Steady-State Assumption

Enzymes Catalysis by ENZYME There are many chemical compounds in the living cell. How they are manufactured and combined at sufficient reaction rates under relatively mild temperature and pressure? How does the cell select exactly which reactants will be combined and which molecule will be decomposed? Catalysis by ENZYME

Enzymes Enzymes are biological catalysts that are protein molecules in nature- react in mild condition They are produced by living cells (animal, plant, and microorganism) and are absolutely essential as catalysts in biochemical reactions. Almost every reaction in a cell requires the presence of a specific enzyme– related to its particular protein structure. A major function of enzymes in a living system is to catalyze the making and breaking of chemical bonds. Therefore, like any other catalysts, they increase the rate of reaction without themselves undergoing permanent chemical changes.

Over 2000 enzymes have been identified Often named by adding the - ‘ase’ to the name of substrate acted upon, or the reaction catalyzed such as urease, alcohol dehydrogenase The majority of cellular reactions are catalyzed by enzymes

Some protein enzyme required a non-protein group for their activity. Cofactors: metal ions, Mg, Zn, Mn, Fe. Coenzyme: complex organic molecule, NAD, FAD, CoA Vitamins Catalyze biochemical reactions breaking, forming and rearranging bonds. Catalytic function – very specific and effective (Specific because of conformational shape) Dictated by the enzyme active site. Some active sites allow for multiple substrates.

Enzymes reduce the activation energy of a reaction Enzymes are catalysts Catalyst: chemical that changes the rate of a reaction without being consumed Recycled (used multiple times) Enzymes reduce the activation energy of a reaction Amount of energy that must be added to get a reaction to proceed

Catalysts A catalyst is unaltered during the course of a reaction and functions in both the forward and reverse directions. In a chemical reaction, a catalyst increases the rate at which the reaction reaches equilibrium. For a reaction to proceed from starting material to product, the chemical transformations of bond-making and bond-breaking require a minimal threshold amount of energy, termed activation energy. Generally, a catalyst serves to lower the activation energy of a particular reaction.

Enzyme Function Enzymes lower the activation energy of reaction catalyzed ( They do this by binding to the substrate of the reaction, and forming an enzyme-substrate (ES) complex) Substrate binds to a specific site on the enzyme called the active site Multi-substrate reactions possible ‘Lock and key’ model

The activation energy for the decomposition of hydrogen peroxide varies depending on the type of catalysis. Type of catalysis Activation energy Uncatalyzed reaction at 20°C 18 kcal/mol Enzymatically catalyzed (catalase) 7 kcal/mol Chemically catalysed (by collodial platinum) 13 kcal/mol Enzyme lower the activation energy of the reaction by binding the substrate and forming an enzymes-substrate complex.

Comparison of activation energies in the uncatalyzed and catalyzed decompositions of ozone.

Important Terms To Remember! active site - a region of an enzyme comprised of different amino acids where catalysis occurs or a small portion of the surface of an enzyme which a specific chemical reaction is catalyzed substrate - the molecule being utilized and/or modified by a particular enzyme at its active site co-factor - organic or inorganic molecules that are required by some enzymes for activity. These include Mg2+, Fe2+, Zn2+ and larger molecules termed co- enzymes like nicotinamide adenine dinucleotide (NAD+), coenzyme A, and many vitamins.

Types of Enzymes holoenzyme - a complete, catalytically active enzyme including all co-factors OR an enzyme containing a non protein group apoenzyme - the protein portion of a holoenzyme minus the co-factors OR the protein part of holoenzyme (holoenzyme = apoenzyme+cofactor) isozyme - (or iso-enzyme) an enzyme that performs the same or similar function of another enzyme that occur in several different molecular forms.

Nomenclature of enzyme Originally enzymes were given non descriptive names such as: rennin : curding of milk to start cheese-making processor pepsin : hydrolyzes proteins at acidic pH trypsin : hydrolyzes proteins at mild alkaline pH The nomenclature was later improved by adding the suffix -ase to the name of the substrate with which the enzyme functions, or to the reaction that is catalyzed, for example:

Alcohol dehydrogenase Glucose isomerase Glucose oxidase Lactic acid dehydrogenase

Enzyme reactions are different from chemical reactions, as follows: 1. An enzyme catalyst is highly specific, and catalyzes only one or a small number of chemical reactions. A great variety of enzymes exist, which can catalyze a very wide range of reactions. 2. The rate of an enzyme-catalyzed reaction is usually much faster than that of the same reaction when directed by nonbiological catalysts at mild reaction condition. 3. A small amount of enzyme is required to produce a desired effect. 4. Enzymes are comparatively sensitive or unstable molecules and require care in their use.

Enzymatic Reaction Principles Biochemically, enzymes are highly specific for their substrates and generally catalyze only one type of reaction at rates thousands and millions times higher than non-enzymatic reactions. Two main principles to remember about enzymes are : they act as CATALYSTS (they are not consumed in a reaction and are regenerated to their starting state) they INCREASE the rate of a reaction towards equilibrium (ratio of substrate to product), but they do not determine the overall equilibrium of a reaction.

Reaction Rates The rate of the reaction is effected by several factors including the concentration of substrate, temperature and pH. For most standard physiological enzymatic reactions, pH and temperature are in a defined environment (eg; pH 6.9-7.4, 37oC). This enzymatic rate relationship has been described mathematically by combining the equilibrium constant, the free energy change and first or second-order rate theory. Keq = e−∆Go/RT The net result for enzymatic reactions is that the lower the activation energy, the faster the reaction rate, and vice versa.

Specificity Most synthetic catalyst are not specific i.e., they will catalyze similar reactions involving many different kinds of reactants. While enzymes are specific. They will catalyze only one reaction involving only certain substances.

Binding Energy The interaction between enzyme and its substrate is usually by weak forces. In most cases, Van der Waals forces and hydrogen bonding are responsible for the formation of ES complexes. The substrate binds to a specific site on the enzyme known as the active site.

Classification of Enzyme Enzymes fall into 6 classes based on function Oxidoreductases: which are involved in oxidation, reduction, and electron or proton transfer reactions Transferases : transfer of functional group Hydrolases : which cleave various covalent bonds by hydrolysis Lyases : catalyse reactions forming or breaking double bonds Isomerases : catalyse isomerisation reactions Ligases : join substituents together covalently.

ENZYME KINETICS

Enzyme Kinetics Mathematical models of single-substrate-enzyme-catalyzed reactions were first developed by Henri in 1902 and Michaelis & Menten in 1913 Simple enzyme kinetics are now commonly referred to as Michaelis-Menten or ‘saturation’ kinetics At high substrate concentrations, all active sites on the enzyme are occupied by substrate – enzyme is saturated Models are based on data from batch reactors with constant liquid volume in which the initial substrate, [S0], and enzyme, [E0], concentrations are known

Enzyme kinetics deals with the rate of enzyme reaction Kinetic studies of enzymatic reactions provide information about : (1)the basic mechanism of the enzyme reaction and (2) other parameters that characterize the properties of the enzyme. The rate equations developed from the kinetic studies can be applied in : (1)calculating reaction time, (2) yields, and (3) optimum economic condition, which are important in the design of an effective bioreactor.

Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E) in a reactor as: If you measure the concentrations of substrate and product with respect to time, the product concentration will increase and reach a maximum value, whereas the substrate concentration will decrease as shown in Figure 2.1

The mechanism of one substrate-enzyme reaction can be expressed as: The rate of reaction can be expressed in terms of either the change of the substrate Cs or the product concentrations CP as follows: change of the substrate Cs product concentrations CP Brown (1902) proposed that an enzyme forms a complex with its substrate. The complex then breaks down to the products and regenerates the free enzyme. The mechanism of one substrate-enzyme reaction can be expressed as:

One of the original theories to account for the formation of the enzyme-substrate complex is the "lock and key" theory. The enzyme represents the lock and substrate represents the key. The main concept of this hypothesis is that there is a topographical, structural compatibility between an enzyme and a substrate which optimally favors the recognition of the substrate as shown in Figure 2.3.

In multi substrate, enzyme-catalyzed reactions, enzymes can hold substrates such that reactive regions of substrates are close to each other and to the enzyme’s active site, which is known as the proximity effects (nearest in distance). Multisubstrate enzyme catalyst reaction

Also, enzymes can hold substrates at certain positions and angles to improve the reaction rate, which is known as the orientation effect. Alteration of active site by activator

The reaction rate equation can be derived from the preceding mechanism based on the following assumptions: The total enzyme concentration stays constant during the reaction, The amount of an enzyme is very small compared to the amount of substrate. Therefore, the formation of the enzyme substrate complex does not significantly deplete the substrate. The product concentration is so low that product inhibition may be considered negligible.

Michaelis - Menten or ‘saturation’ kinetics

Single-Substrate Enzyme Kinetics It is assumed that: The ES complex is established very rapidly 2) The rate of the reverse reaction of the second step is negligible (i.e k-2~0) (Assumption 2 is typically only valid when product (P) accumulation is negligible, at the beginning of the reaction) Enzyme Enzyme Substrate Complex Product Substrate

This model are based on data from batch reactors with constant liquid volume in which the initial substrate,[S0], and enzyme [E0], concentration are known. An enzyme solution has a fixed number of active sites to which substrate can bind. At high substrate concentrations, all these sites may be occupied by substrates or the enzyme is saturated. Two major approaches used in developing a rate expression for enzyme catalyzed reactions are , rapid-equilibrium approach and (2) quasi-steady-state approach.

Rate of product formation: Rate of variation of the ES complex: Since the enzyme is not consumed, the conservation equation yields, At this point, an assumption is required in order to achieve an analytical solution

Assumption Rapid-Equilibrium Assumption (2) Quasi-Steady-State Assumption

The Rapid Equilibrium Assumption Henri and Michaelis and Menten used essentially this approach. Assuming equilibrium in the first part of the reaction (E+S forms ES), we can use the equilibrium coefficient to express [ES] in terms of [S] The equilibrium constant is: Since , if the enzyme is conserved, then

Substitution in Yields, Where, Michaelis-Menten constant (the prime (‘) indicates that it was derived assuming rapid equilibrium) Low value: enzyme has high affinity for the substrate Corresponds to the substrate concentration, giving half-maximal reaction velocity. Vm= maximum forward rate of the reaction (change with the addition of additional enzyme but not addition of substrate)

Rate of Reaction as a Function of Substrate Concentration

Quasi-Steady-State Assumption Briggs and Haldane first proposed Quasi-steady-state assumption In a batch reactor at closed system, [E0] is considered very small compared S Therefore, d(ES)/dt ≈0 From equation ----1 ----2

----3 ----4 ----5 Substituting , , and solve equation 2 for [ES], Production formation kinetics, Substitute equation 3 into 4, ----3 ----4 ----5

Substituting, There is difference between Michaelis-Menten constant [K’m=k-1/k1] and Quasi-steady- state constant [K’m=k-1+k2/k1] . ----6

Determination of Rate Parameters for Michaelis-Menten Type Kinetics Lineweaver-Burk plot Eadie- Hofstee plot Hanes-Woolf plot Batch kinetics

1. Lineweaver-Burk Plot From equation 6 (Quasi-steady-state ), Double reciprocal plot slope Y-intercept Lineweaver-Burk plot gives good estimates on Vm but not necessarily on Km (error relates with substrate conc)

2. Eadie–Hofstee Plot From equation 6, Rearranged equation 6, plot v versus v/[S] gives a line of slope –Km and y-axis intercept of Vm Can be subject to large errors since both coordinates contain v, but less bias on points at low [S]

3. Hanes–Woolf plot Rearrangement of equation 6 yields, slope intercept This plot is used to determine Vm more accurately.

4. Batch kinetics Integration of equation 6 and rearranged yields, slope Y-intercept

Let’s Understand it More based on your first Experiment ERT 317

Experiment 1 Effect of Substrate Concentration on Enzyme Kinetics Study OBJECTIVES To develop a suitable standard curve for enzyme assay To analyze the effect of substrate concentration on the activity of enzyme To determine Vmax and Km from the enzyme reaction using enzyme kinetics plots.

COURSE OUTCOMES CO1 – Ability to develop enzyme reactions based on its kinetics study and applied catalysis INTRODUCTION The enzyme α-amylase can catalyze the hydrolysis of internal α -1,4-glycosidic bond present in starch with the production of reducing sugars. In the study of substrate concentration on enzyme kinetics, the enzyme is kept constant where as the concentration of starch is taken in increasing order. As the substrate concentration increases, the amount of products produced in every successive tube also increases. This enzyme-substrate reaction can be determined by measuring the increase in reducing sugars using the 3,5-dinitrosalicylic acid reagent. In an alkaline condition, the pale yellow coloured the 3,5-dinitrosalicylic acid undergo reduction to yield orange coloured 3-amino-5-nitrosalicylic acid. The absorbance of resultant solutions is read at 540nm. The intensity of colour depends on the concentration of reducing sugars produced.

Starch + α-amylase Maltose + glucose hydrolysis Starch + α-amylase Maltose + glucose Amylose comprises 15-30% of the common starches. Amylose is a linear polymer containing up to 6000 glucose units, connected by α (1, 4) linkages. Therefore, we use α-amylase to hydrolyzed starch in this hydrolysis process. The hydrolysis of starch with a low molecular weight, catalyzed by an α- amylase, is one of the most important commercial enzyme processes.

What does alpha amylase do? hydrolysis Starch + α-amylase Maltose + glucose What does alpha amylase do? α-Amylase is a protein enzyme EC 3.2.1.1 that hydrolyses alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. The individual subunits that make up maltose are glucose

Starch + α-amylase Maltose + glucose hydrolysis Starch + α-amylase Maltose + glucose Your Task: 1) Develop of maltose standard curve why? How? -to measure the product of reducing sugar (after hydrolysis process), we NEED a standard. Pure Sugar : Maltose (main reducing sugar produced from starch after enzymatic hydrolysis) Reagent needed: DNS reagent (the intensity of colour in DNS depends on the concentration of reducing sugars produced)

Simple Method: Prepare at least 5 different concentration of pure sugar (Maltose) Prepare 1 Blank (no sugar/no maltose) All samples + DNS reagent (follow DNS method) Read absorbance @ 540 nm (for blank: Zeroing) Plot Absorbance reading (y-axis) VS Maltose conc (x-axis) Linear graph with an equation Y=mx+c (make sure R2 for any standard curve is 0.999)

Your Task: 2) Study effect of substrate concentration on enzyme activity -Vary substrate (starch) concentration but fix the enzyme concentration. SIMPLE METHOD: Run the hydrolysis process (different starch conc react with an enzyme) at 37oC, 3 min. Produced product (hydrolysate) containing reducing sugars (maltose) Blank: no starch All product samples contain unknown conc of maltose. Therefore, we need to run DNS method. Samples + DNS reagent (follow DNS method) . Cek concentration of samples using MALTOSE Standard Curved.

Starch concentration, [S] (%) Abs at 540 nm Amount of maltose (µg) Velocity [V] in µmoles/min 1/[V] 1/[S] 0.02 0.04 0.06 0.08 0.10 Draw the Michaelis-Menten’s plot and Line Weaver Burk plot using the data in Table 1.3 and find the Vmax and Km from the plot. Compare the value of Vmax and Km from both plot and give your reason of their differences.

Michaelis-Menten’s plot

Lineweaver-Burk Plot From equation 6 (Quasi-steady-state ), Double reciprocal plot slope Y-intercept Lineweaver-Burk plot gives good estimates on Vm but not necessarily on Km (error relates with substrate conc)

Thank You