CHAPTER 2

ENZYME KINETICS & INDUSTRIAL ENZYMOLOGY

In this topic, you will learn about:1) Nature of enzyme action.2) Michaelis-Menten Equation, Lineweaver-Burk, & other plots.3) Steady state approach.4) Enzyme inhibition; competitive & non-competitive inhibition.5) Temp & pH effect.

1) NATURE OF ENZYME ACTION

DEFINITION of ENZYMES

Biological catalysts that are protein molecules in nature. Produced by living cells (animal, plant & microorganism). Absolutely essential as catalysts in biochemical reactions.

FUNCTION of ENZYMES

To catalyze the making and breaking of chemical bonds.To increase the rate of reaction without themselves undergoing permanent chemical changes.

ENZYME REACTIONS

ENERGY OF ACTIVATION

• In a chemical reaction, the reactants must absorb a certain amount of energy from the environment before a reaction can take place. • The specific amount of energy required for the reaction to proceed is called the ENERGY OF ACTIVATION.

ENERGY OF ACTIVATION

• In reactions occurring outside of living organisms, simply adding heat can provide activation energy.• Inside of living organisms, however, another method must be used. Heat is very harmful to the cells and proteins of plants and animals.

ENERGY OF ACTIVATION

ENERGY OF ACTIVATION

• To overcome the activation energy required for certain reactions to take place, living organisms employ enzymes. Enzymes function by being able to LOWER the activation energy needed in specific reactions. • With a lower energy requirement, more molecules will be able to react with each other and the reaction can swiftly occur at temperatures able to support life.

ENERGY OF ACTIVATION

ENZYME REACTIONS vs

CHEMICAL REACTIONS Highly specific - catalyze only one or small number of chemical reactions Their rate of reaction is usually much faster than non-biological catalyst The reaction conditions ( T, P, pH, and so on) are very mild. Enzymes are comparatively sensitive or unstable molecules and require care in their use.

NOMENCLATURE of ENZYMES*Non-descriptive name such as:rennin curding of milk to start cheese-making processerpepsin hydrolyzes proteins at acidic pHtrypsin hydrolyzes proteins at mild alkaline pH

NOMENCLATURE of ENZYMES*Name of substrate + ase :Substrate Enzyme ProductStarch α-amylase glucose + maltose + oligosaccharideslactose lactase glucose + galactosefat lipase fatty acids + glycerolmaltose maltase glucoseurea + H2O urease 2NH3 + CO2cellobiose cellobiase glucose

NOMENCLATURE of ENZYMES*Reaction which is catalyzed + ase :Enzymes Reactionalcohol dehydrogenase ethanol + NAD+ ↔ acetaldehyde + NADH2glucose isomerase glucose ↔ fructoseglucose oxidase D-glucose + O2 + H2O → gluconic acidlactic acid dehydrogenase lactic acid → pyruvic acid

COMMERCIAL APPLICATION of ENZYMES

Industrial enzymes amylase, protease, glucose isomerase, lipase, catalase, penicilin acylasesAnalytical Enzymes glucose oxidase, galactose oxidase, alcohol dehydrogenase, hexokinase, muramidase, cholesterol oxidaseMedical Enzymes asparaginase, proteases, lipases, streptokinase* what is HFCS ( high-fructose corn syrup)? what kind of enzymes contribute in the process of HFCS?

ENZYME KINETICS

• Study of the rates of enzyme-catalyzed reactions• Provides information on enzyme specificities and mechanisms

KINETICS?????

ENZYME ACTIONLOCK AND KEY MODEL• An enzyme binds a substrate in a region called the active site.• Only certain substrates can fit the active site.• Amino acid R groups in the active site help substrate bind.• Enzyme-substrate complex forms.• Substrate reacts to form product.• Product is released.

ENZYME ACTIONLOCK AND KEY MODEL

ENZYME ACTIONLOCK AND KEY MODEL

ENZYME ACTIONINDUCED FIT MODEL• Enzyme structure flexible, not rigid.• Enzyme and flexible active site adjust shape to bind substrate.• This sudden change in shape can lead to the breaking of bonds within a single substrate molecule, forming two new molecules. Conversely, it can also bring two-substrate molecules close enough together for them to bond with each other, forming one new molecule.• Increases range of substrate specificity.• Shape changes also improve catalysis during reaction.

ENZYME ACTIONINDUCED FIT MODEL

ENZYME ACTIONINDUCED FIT MODEL

ENZYME ACTIONINDUCED FIT MODEL

FORMULA FOR A SIMPLE ENZYME-CATALYZED

REACTION

E = free enzyme S = substrate ES = enzyme-substrate complex P = product

FORMULA FOR A SIMPLE ENZYME-CATALYZED

REACTION

2) MICHAELIS- MENTEN EQUATION, LINEWEAVER BURK & OTHER PLOTS

• Leonor Michaelis (1913): noticed that at constant [enzyme] the rate of a reaction increases with increasing [S] until a “maximal velocity” (Vmax) is achieved• This “saturation effect” is an important distinction versus uncatalyzed reactions• Interpretation of data: ES complexes formed until substrate saturation occurs at which point no more substrate binding sites (i.e., enzyme molecules) are available

MICHAELIS-MENTEN EQUATION

MICHAELIS-MENTEN EQUATION

Michaelis and Menten (1913): It is assumed that the product-releasing step (eqn above with k2) is much slower than the reversible reaction (k1 and k-1). The slow step determines the rate, while the other is at equilibrium.

SKS

m max

MICHAELIS-MENTEN EQUATION

MICHAELIS-MENTEN EQUATION

MICHAELIS-MENTEN EQUATION

• Km has a unit of concentration. It is a constant for every enzyme under specific conditions• When [S] <<< Km, Vo is directly proportional to [S]• When [S] >>> Km, Vo = Vmax• When [S] = Km, Vo = ½ Vmax• The Km value of an enzyme indicates the concentration of the substrate required for significant catalysis• Since Km = (k2+k-1)/k1, when k-1 >>> k2, Km = k-1/k1• Dissociation constant of ES, KES = [E][S] / [ES] = k-1/k1• When k-1 >>> k2, Km = KES. High Km = high dissociation = weak binding between E and S• When [E]T = [ES], Vmax = k2[E]T. k2 is called the turnover number: the rate when enzyme is saturated with substrate

SIGNIFICANCE OF MICHAELIS-MENTEN EQUATION

LINEWEAVER-BURK PLOT

• Also called the double-reciprocal plot• 1/Vo = (Km/Vmax).(1/[S]) + 1/Vmax• This is equation for a straight line y = mx + c• Slope = Km/Vmax• Y-intercept = 1/Vmax• X-intercept = -1/Km• Useful for experimental determination of Km and Vmax

LINEWEAVER-BURK PLOT

LINEWEAVER-BURK PLOT

Km values for various enzyme

3) STEADY STATE APPROACH

• Enzymes are required in minute quantities and enhance reaction rates by 1010 to 1020fold• When the enzyme is part of a crude preparation, its concentration is in terms of units• Enzyme activity is the ability of an enzyme to modify a reactant. 1 unit (U) is the enzyme activity that converts 1 µmole of reactant per min under standard conditions.• The specific activity of an enzyme is defined as the activity per unit of mass or U/mg protein

ENZYME ACTIVITY

4) ENZYME INHIBITION: COMPETITIVE & NON-COMPETITIVE INHIBITION

ENZYME INHIBITION

Inhibitors • Cause a loss of catalytic activity.• Change the protein structure of an enzyme.• May be a reversible or irreversible inhibition. • Specific enzyme inhibitors regulate enzyme activity and help us understand mechanism of enzyme action. (Denaturing agents are not inhibitors).

Inhibitors • Irreversible inhibitors form covalent or very tight permanent bonds with the active site of the enzyme and incapacitating the enzyme. 3 classes: group-specific reagents, substrate analogs, suicide inhibitors.

ENZYME INHIBITION

Inhibitors • Reversible inhibitors form an EI complex that can be dissociated back to enzyme and free inhibitor. 3 groups based on their mechanism of action:competitive, non-competitive and uncompetitive.

ENZYME INHIBITION

COMPETITIVE INHIBITIONA competitive inhibitor• Has a structure similar to substrate• Occupies active site• Competes with substrate for binding to enzyme (active site)• E + S = ES or E + I = EI . Both S and I cannot bind enzyme at the same time• In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES.• This can be reversed / corrected by increasing [S]• Vmax is not changed, Km is increased

NON-COMPETITIVE INHIBITION

A noncompetitive inhibitor• Inhibitor binding site is distinct from substrate binding site, it does not have a structure like substrate• Binds to the enzyme but not active site• Changes the shape of enzyme and active site• Substrate cannot fit altered active site• Can bind to free enzyme E and to ES• E + I = EI, ES + I = ESI or EI + S = ESI

NON-COMPETITIVE INHIBITION

A noncompetitive inhibitor• Both EI and ESI are enzymatically inactive• The effective functional [E] (and [S]) is reduced• Reaction of unaffected ES proceeds normally• Inhibition cannot be reversed by increasing [S]• Km is not changed, Vmax is decreased

5) TEMPERATURE & pH EFFECT

The study of enzyme reaction rates is called enzyme kinetics. Enzyme kinetics are affected by:•Temperature and pH: Each enzymatic reaction has an optimum pH and optimum temperature. Extreme temperature or pH disrupts enzyme structure and therefore reaction rate•Substrate concentration: The reaction rate = k [P] / [S]. The rate can be increased by adding more substrate, or by removing product as it is formed

FACTOR AFFECTING REACTION RATE

• Little activity at low temperature• Rate increases with temperature• Most active at optimum temperatures (usually 37°C in humans)• Activity lost with denaturation at high temperatures

FACTOR AFFECTING ENZYME ACTIONTEMPERATURE

FACTOR AFFECTING ENZYME ACTIONTEMPERATURE

FACTOR AFFECTING ENZYME ACTIONTEMPERATUREFACTOR AFFECTING ENZYME ACTIONTEMPERATURE

• Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant).• Maximum activity reached when all of enzyme combines with substrate

FACTOR AFFECTING ENZYME ACTIONSUBSTRATE CONCENTRATION

FACTOR AFFECTING ENZYME ACTIONSUBSTRATE CONCENTRATION

• Maximum activity at optimum pH• R groups of amino acids have proper charge• Narrow range of activity• Most lose activity in low or high pH

FACTOR AFFECTING ENZYME ACTIONpH

FACTOR AFFECTING ENZYME ACTIONpH

END OF CHAPTER 2