Enzymology Introduction Flashcards by pattie o.

How enzymes act in reactions

Lower activation energy for reactions

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Where enzymes are found

In all body tissues, appear in serum after cellular injury

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Enzyme specificity - absolute specificity

Combines with one substrate and catalyzes one reaction

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Enzyme specificity - group specificity

Combines with substrates containing a particular chemical group

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Enzyme specificity - bond or reaction specificity

Forms or breaks one type of covalent bond

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Enzyme specificity - stereoisomeric/geometric specificity

Combines with only one optical or geometric isomer

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Main function of enzymes

Accelerates biochemical reactions; biological catalyst

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Enzyme involvement in reactions

Enzyme is not a direct participant and does not change

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Binding sites of enzymes

Active site (main), Allosteric site (for cofactors)

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Role of cofactors

Activate enzyme; reaction won’t proceed without them

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Types of cofactors

Activator (inorganic), Coenzyme (organic)

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Apoenzyme + Cofactor

Holoenzyme

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Inactive form of enzyme

Proenzymes/Zymogens

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Common enzymes with proenzymes

Hydrolases

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Enzyme classification EC code 1

Oxidoreductases - Redox reaction (hydrogen and oxygen)

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Function of oxidoreductases

Addition of hydrogen and oxygen; oxidation removes hydrogen, adding oxygen replaces hydrogen

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Examples of oxidoreductases

Dehydrogenase, oxidase, hydrogenase, oxygenase

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Enzyme classification EC code 2

Transferases - Transfers functional group (not H and O)

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Function of transferases

Transfers functional groups like carbohydrate, phosphate, hydroxy group, amino acid

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Example of transferase reaction

Creatine kinase + ATP <-> CP + ADP

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Examples of transferases

Transferase, kinase (phosphate extraction from ATP), phosphorylase (transfers phosphate)

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Enzyme classification EC code 3

Hydrolases - Removes functional group with water

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Examples of hydrolases

Amylase (destroys glycosidic bonds), Lipase (destroys ester bonds), Trypsin/Chymotrypsin (destroys peptide bonds)

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Enzyme classification EC code 4

Lyases - Removes functional group without water (H2O)

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Function of lyases

Products have double bond

Examples of lyases

Aldolase, decarboxylase

Enzyme classification EC code 5

Isomerases - Converts isomer from one orientation to another

Example of isomerase reaction

D to L orientation of glucose

Identification of isomerases

Isomerase in name; Example - triose phosphate isomerase

Enzyme classification EC code 6

Ligases - Bond formation coupled with ATP hydrolysis

Example of ligase

Glutathione synthetase

Enzyme kinetics concept

Substrate needs specific energy to proceed with a reaction, called activation energy

Enzyme's role in reaction

Enzyme reduces energy barrier, accelerates reaction

Equilibrium in enzyme kinetics

Same result, faster reaction

Effect of heat on enzyme reactions

Heat accelerates reactions by increasing molecular movement

Enzyme kinetics reaction mechanism

Enzyme + Substrate <-> ES <-> Product + Enzyme (free again)

Zero order kinetics

Enzyme < Substrate, rate is constant, enzyme becomes rate-limiting when active site is saturated

First order kinetics

Enzyme > Substrate, rate is dependent on substrate concentration, increased substrate increases enzyme-substrate complex

Rate limiting component in zero order kinetics

Enzyme

Rate limiting component in first order kinetics

Substrate

Inhibitor type same structure as substrate, binds to active site, reversible

Competitive inhibitor:

Inhibitor type Binds to enzyme (not active site), reversible, may destroy structure

Non-competitive inhibitor:

Inhibitor type Binds to ES complex, increasing substrate concentration, no product formation

Uncompetitive inhibitor:

Inhibitor type can bind to either substrate or enzyme

Mixed inhibitor:

Michaelis-Menten equation

v = Vmax (S) / Km + (S)

Lineweaver-Burke plot

Double reciprocal plot of Michaelis-Menten equation: y = mx + b

Fixed-time/End point measurement

One-time measurement with stop solution (weak acid)

Kinetic/Continuous measurement

Multiple measurements, checks actual activity and monitors linearity

Measurement unit for enzyme activity

IU: Amount of enzyme that catalyzes 1 umol of substrate

Measurement unit for enzyme concentration

IU/L

SI unit for enzyme activity

Katal: Number of moles per second (mol/sec)

Conversion between IU and Katal

1 IU = 17 katals

Conventional unit for enzyme activity

IU: Amount of enzyme that catalyzes 1 umol of substrate per minute

SI unit for enzyme activity

Katal: Amount of enzyme that catalyzes 1 mol of substrate per second

Conversion between IU and nkat

1 IU = 0.0161 ukat or 16.7 (17) nkat

Conversion between IU and kat

1 kat = 6 x 10^7 IU

Reactants combined, reaction proceeds for set time, reaction stopped, amount of reaction measured

Fixed-time endpoint method

Multiple measurements of absorbance over time using a spectrophotometer

Continuous-monitoring kinetic method

Factor affecting enzymatic reaction, pH range for most reactions

7.0-8.0 (except ACP: 4.5, ALP: 9-10)

Effect of temperature on enzymatic reaction rate

Rate doubles for every 10°C increase

Temperature range for denaturation in enzymatic reactions

40-50°C

Incubation temperature deviation in enzyme assays

±0.1°C

Definition of cofactors

Nonprotein entities that bind to enzymes for reactions

Activators as cofactors

Inorganic, metallic or non-metallic ions (e.g. Zn, Ca, Mg, Cl)

Definition of coenzymes

Organic substances, serve as second substrates in enzymatic reactions

Coenzymes bound tightly to enzymes

Prosthetic groups

Example of coenzyme

NAD(P) for dehydrogenases, Pyridoxal PO4 for AST, ALT

Effect of adding substrate in competitive inhibitor

Increased substrate reverses inhibition

Effect of adding substrate in non-competitive inhibitor

No effect

Effect of adding substrate in uncompetitive inhibitor

Increases inhibition

Lineweaver-Burk plot in competitive inhibitor

Same Cmax, increased Km (rightward shift)

Lineweaver-Burk plot in non-competitive inhibitor

Decreased Vmax, same Km

Lineweaver-Burk plot in uncompetitive inhibitor

Decreased Vmax, decreased Km

Macroenzyme definition

High-molecular-mass forms of serum enzymes bound to immunoglobulin or non-immunoglobulin substance

Examples of enzymes with macroforms

CK, LD, ALT, AST, GGT, ACP, ALP, AMS, LPS