Enzyme Catalysis

BIOCHEMICAL ENGINEERING INTRODUCTION TO ENZYME KINETICS

STRUCTURE 

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GLOBULAR PROTEIN ( 3-D STRUCTURE) PRIMARY STRUCTURE SECONDARY STRUCTURE TERTIARY STRUCTURE QUATERNARY STRUCTURE ISOENZYMES, ISOZYMES, ISOFORMS

TYPES AND NOMENCLATURE  

TRIVIAL NAME: ‘-ase’ ENZYME COMMISSION NUMBER(EC)

UNIQUENESS    

High specificity for substrate. Operate under ‘mild’ conditions High reaction rates. Can be regulated.

Progress of a chemical reaction Free Energy (G)

transition state

Activation energy

initial state

∆G

∆G is negative; reaction goes

final state

Progress of reaction

Progress of Enzyme reaction S to P Enzyme lowers activation energy

Free Energy (G)

transition state

Activation energy

S

initial state

ES

P

∆G

∆G is negative; reaction goes

final state

Progress of reaction

How do catalysts work? Activation Energy

Catalysts work by stabilizing the transition state of a chemical reaction, which lowers the activation energy of the reaction

What sort of rate acceleration can enzyme provide? Consider the reaction:

No Catalyst Pt black Enzymes

Eact Relative Rate 18 kcal/mol 1 12 kcal/mol 1X10^4 2 kcal/mol 3X10^11

Enzymes are capable of providing astonishing rate enhancements

Lets take a look at a real example! ATP

Mg(2+)

ATP hydrolysis as an example

Does an enzyme only catalyze the forward reaction? NO!

Because the free energy difference between reactants and products of a reaction and the starting concentration of each determines the direction.

How do enzymes do the amazing things they do?

UNIQUE FEATURES OF THE ENZYMES 

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Enzymes increase rate of reaction but DO NOT change equilibrium point. Enzymes DO NOT supply energy to reaction, but instead lower the activation energy requirement Substrate(s) bind at ACTIVE SITE(S) with BINDING SITE and CATALYTIC GROUPS

NOVEL USES OF ENZYMES 

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as monitors of toxic chemical levels in food and water exploitation of enzymes as electrocatalysts (specific biosensors) Enzyme utilisation in formation of food flavours and aroma compounds Enzyme technology in the prevention of dental cavities

APPLICATIONS OF ENZYMES 

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Proteas: Clotting and manufacture of cheese Glucose Isomerase: Manufacture of high-fructose syrups as 'high sweeteners' Glucose oxidase : Analysis of blood glucose levels Pectinases: Juice/Wine clarification Amylases: Brewing

ENZYME SUBSTRATE BINDING AND SPECIFICITY 

COMPLEMENTARITY OF STRUCTURES

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STERIC, CHARGE NEUTRALITY AND HYDROGEN BOND FACTORS

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ATP TO MYOSIN, Kd = 10-13 M

MECHANISM OF CATALYSIS 

Induced fit model.

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Catalysis by bond strain

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Catalysis by proximity and orientation

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Catalysis Involving Proton donors or acceptors

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Covalent Catalysis

ENZYME ASSAYS 

Spectrophotometri c or radiometric

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Initial rate over 1 min

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Rapid assays

FAST REACTIONS 

Pre-steady state phase less than 1s

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Rapid mixing

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Estimation of unitary rate constants

STOPPED FLOW

CONTINUOUS FLOW ULTRA FAST MIXING

CONTINUOUS FLOW QUENCH FLOW

RELAXATION METHOD 

Reaction at equilibrium perturbed

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Spectroscopic monitoring

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Temperature, Pressure and electric field strength

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Reactions with half lives 10-10 s

Michaelis-Menten Equation 

Briggs-Haldane equation

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Vmax = K2 [Eo]

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M-M Equation : K2 << K

-1

Solution of MM Equation 1st Approximation 

[E] is small

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[A] = [A]o - [C]

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2

nd

Approximation

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[E] = [E]0 - [B]

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[A] = [A]0

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Multi Substrate Reactions 

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Modified M-M eq Types: Sequential Reactions  Ping-Pong Reactions 

Measurement of Vi     

Set up a series of tubes containing graded concentrations of substrate, [S]. At time t=0, add a fixed amount of the enzyme preparation. Over the next few minutes, measure the concentration of product formed. If the product absorbs light, we can easily do this in a spectrophotometer. Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observe is the initial velocity Vi.

Michaelis-Menten plot 

Equation: V =(Vmax [S]) / (Km + [S])

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Rectangular hyperbola Hard to achieve Vmax Hard to work out inhibition patterns

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Convert hyperbolic curve to a straight line by doing a reciprocal plot – Lineweaver-Burk

Lineweaver-Burk OR Double Reciprocal plot 

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Plot of 1/v against 1/[S] Y-intercept: vmax

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X-intercept: Km

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Better estimate of vmax

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Also useful in determining the nature of inhibition

1 1 Km 1 = + v v max v max [ S ]

Other plots •

Eadie-Hofstee plot: Plot of v against v/[S]. Y-intercept: vmax

•

X-intercept: Km

Km v = v max − v [S ]

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Hanes-Wolf plot: Plot of [S]/v against [S]. X-intercept: Km

[ S ] [ S ] Km = + v v max v max

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• •

3. Y-intercept: vmax

Case Study: Characterization of ALP enzyme activity

Spectrophotometer: (PNP absorbs at 410 nm) 

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absorbance value recorded every second (Lab view) absorbance (A)  concentration (mM) 3 ml total volume: PNPP + buffer (blank) , ALP added plot absorbance vs. time

Determine Activity of Alkaline Phosphatase: 

0.75 ml 0.4 mM PNPP 0.2

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M Tris-HCl buffer added (3 ml total volume)

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0.2, 0.3, 0.4, 0.5 ml ALP

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initial slope A vs. time plot activity (mU/ml)

Michaelis-Menten Modeling:  0.075, 0.40, 0.75, 1.15, 1.50 ml 0.4 mM PNPP  buffer added (3 ml total volume)  0.2 ml ALP  initial slope A vs. time  reaction velocity (mM/min) Absorbance

0.400 mL Enzyme + 0.75 mL 0.4 mM Substrate 1.4 1.2

y = 0.00472x + 0.04006 R2 = 0.99986

1 0.8 0.6 0.4 0.2 0 0

100

200 Time (s)

300

400

Lineweaver-Burk plot 1/v (min/mM) vs. 1/[S] (mM-1) 

x-intercept = -308.951 mM-1 = -1/Km

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Km = 0.00337 mM

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y-intercept = 100.24 min/mM = 1/Vmax

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1/V (min/mM)

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y = 0.3374x + 100.24 R2 = 0.9586

Lineweaver-Burke Plot

-400

-300

-200

Vmax = 0.00999 mM/min

-100

160 140 120 100 80 60 40 20 0 -20 0 -40

100

1/[s] (1/mM)

-1/Km

1/Vmax

200

Eadie-Hofstee v/[S] (min-1) vs. v (mM/min) 

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Vmax = 0.010045 mM/min y-intercept = Vmax/Km

2.5 2 1.5 1 0.5 0 0

0.005

= 2.7575 min-1 

Km = 0.00365 mM

y = -274.51x + 2.7575 2 R = 0.938

3

x-intercept = Vmax = 0.010045 mM/min

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Eadie-Hofstee Plot

v/[S] (1/min)

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0.01

0.015

v (mM/min)

Vmax/Km

Vmax

Hanes-Wolf Plot [S]/v (min) vs. [S] (mM) 

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x-intercept = -0.00550 mM = -Km Km = 0.00550 mM

y = 97.434x + 0.536 R2 = 0.9995

Hanes-Woolf Plot 25 20 15 [S]/v (min)

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10 5 0

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y-intercept = 0.536 = Km/Vmax Vmax = 0.01026 mM/min

-0.05

0

0.05

0.1

-5 [S] mM

-Km

Km/Vmax

0.15

0.2

0.25

Conclusions 

Hanes-Wolf Model best linearizes Michaelis-Menten equation 

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Lineweaver-Burk plot does not equally weight all data points  

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greatest linear correlation (R2=.9995 )

exaggerates error at low substrate levels where measurements are less accurate (slower reaction velocities) Another disadvantage is that most experimental measurements involve relatively high [S] and are thus crowded toward the left side of the plot.

The Eadie-Hofstee plot not as error-prone as Lineweaver-Burke plot  

equal weighting to all data points

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Application of Km: Hexokinase and Glucokinase: Catalyze the first step in glucose metabolism:

Glucose + ATP = Glucose-6-phosphate + ADP Normal [ATP] = 1-2 mM. Hence reaction is independent of [ATP]. Hexokinase: found in cells utilizing glucose as an energy source. Low Km for glucose = 0.1 umol/L so that it will be saturated at lower glucose levels and inhibited allosterically by G-6-P product. provides G-6-P for energy production • •

Intracellular glucose levels = 0.2 umol/L. Conversely, blood glucose levels = 5 mmol/L, therefore, hexokinase would not operate efficiently in that environment at those higher levels.

Application of Km: Hexokinase and Glucokinase:  Glucokinase – present in liver hepatocytes and  

pancreatic cells and plays a physiologic role in glucose synthesis and storage which takes place in the liver. Provides G-6-P for glycogen synthesis The liver is responsive to changes in blood glucose concentration 

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Catalyzes the same reaction but has an > Km for glucose = 10 mmol/L and is not inhibited by the product. Km glucokinase > Km for hexokinase. 

Glucokinase forms its product only when glucose levels become higher such as following a meal.

Application of Km: Hexokinase and Glucokinase:

Multisubstrate Reaction Kinetics 

Reactions with more than one substrate, of the A + Btype →C+D

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Three mechanisms characterized by the order of substrate addition to the enzyme and the order of product release. Reaction equations differ depending on mechanisms r max[ A][ B]

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r=

( Kma + [ A])( Kmb + [ B ])

Multisubstrate Reactions 

Three mechanisms:

5.

Sequential order: Substrate addition and products release both in obligate order.

2. Random order: Substrate addition and product release is in random order.

3. Ping-Pong:  The enzyme binds the first S and releases the first P before addition of the second S  Part of the first S is transferred to E to form a modified form F  F now binds the second S and forms the second P  The chemical transformation on F is transferred to second S  E is regenerated

Multisubstrate Reaction Kinetics 

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Sequential kinetics can be distinguished from ping-pong kinetics by initial rate studies. In practice, measure initial rates as a function of one substrate while holding the other constant. Then, vary the concentration of the second substrate and repeat. Lineweaver-Burk analysis should yield a family of lines that intersect at the left of the y-axis of the graph.

Multisubstrate Reaction Kinetics

What is Enzyme Inhibition? 

Interference with the enzyme’s ability to convert substrate to product - usually a decrease in velocity OR a complete cessation of activity.

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Inhibitors bind to the enzyme or enzyme-substrate complex and decrease the enzyme activity.

Why is Enzyme inhibition important? 

Benefits Medicine/drugs  Regulation of processes in human body 

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The other side 

Poisons ( Mustard gas, nerve gas etc)

Types of Inhibition  

Irreversible (suicide) inhibition Reversible inhibition:  Competitive  Non-competitive  Uncompetitive  Mixed

Types of Inhibition Competitive Inhibition

Non Competitive Inhibition

Relevant Examples of Enzyme Inhibition 1)Application in Medicine 

Drug strategy to attack deadly HIV virus.

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HIV virus-attacks Helper T cells and destroys immune system.

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Reverse transcriptase enzyme catalyses production of DNA from

Mechanism of destruction of immune cells by HIV virus

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Enzyme reverse transcriptase is a major target target site for HIV-fighting drugs.

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Zidovudine (AZT), inhibits reverse transcriptase and hence in used in HIV drugs .

More examples of enzyme inhibition 

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Ethanol metabolism in body EthanolAcetaldehydeAcetic acid Disulfiram(Antabuse) drug inhibits aldehyde oxidase. 

Accumulation of acetaldehde.

Respiration Regulation 

Respiration is regulated by feedback inhibtion

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GlucoseCarbon dioxide +water+ATP.

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Citrase synthase is inhibited by ATP.

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Required amount of energy is generated.

Allosteric Enzymes 

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Class of enzymes that bind small, physiologically important molecules and modulate activity. The binding molecules—effectors. Effectors bind to enzyme at distinct site, change is transmitted to catalytic site. Positive and negative effectors.

EFFECT OF VAROIUS PARAMETERS ON ENZYME ACTIVITY   

pH Temperature Solvent

pH 

Affects the activity, structural stability and solubility of the enzyme.

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Only ionic species of a specific charge causes activity of enzyme.

-- A

-- B

pH optimum = (pKa1 + pKa2) / 2

pH for optimum activity •Lipase (pancreas)

- 8.0

•Lipase (stomach)

- 4.0-5.0

•Invertase

- 4.5

TEMPERATURE EFFECT 

Energetic collisions.

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Number of collisions per unit time.

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Unfolding.

On reducing temperature, activity can be restored only to SOME extent.

DEPENDENCE ON SOLVENT 

Enzymes work in aqueous solution.

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In organic phase, UNFOLDING occurs causing deactivation of enzyme.