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Enzyme Catalysis & BiotechnologyEnzyme Catalysis & Biotechnology

Bovine Pancreatic RNase A

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Biochemistry, Life, and all that

BiochemistryBiochemistry - studies biological (living) species in chemical terms- compositions and structure of biochemical molecules, trying to understand their

functions at a molecular level.

General observations on biochemical molecules- generally very large: molecular weight ~ O(1,000) – O(1,000,000) amu

- generally rather simple composition: mostly C, O, H as their main building blocks and relatively few other atoms (such N, P, S) in their functional groups

- generally rather complex structures: specific structure is crucial to their highly specialized functions in biological processes.

All life processes on earth require energy (processes of bio-molecule synthesis are endothermic), which is obtained indirectly from solar energy through plant photosynthesis.

A brief word about biochemistry……traditionally, chemical engineers used organic and inorganic chemistry (along with some physics, lots of math, and other fancy things) to “make stuff”. Anything “bio” was regarded as beyond the reach of industrial activities and/or sufficiently uninteresting. This has changed completely in the past few decades.This has changed completely in the past few decades.

L28L28--33

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Photosynthesis and RespirationPhotosynthesisPhotosynthesis and respirationrespiration as complementary processes in the living world. Photosynthesis uses the energy of sunlight to produce sugars and other organic molecules. These molecules serve as food for other organisms. Many of these organisms carry out respiration, a process that uses O2 to form CO2. In the process, the organisms that respire obtain the chemical bond energy that they need to survive. The first cells on the earth are thought to have been capable of neither photosynthesis nor respiration. However, photosynthesis must have preceded respiration on the earth, since there is strong evidence that billions of years of photosynthesis were required before O2 had been released in sufficient quantity to create an atmosphere rich in this gas. (The earth's atmosphere presently contains 20% O2.)

L28L28--44

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Maintaining Order…Cells must maintain highly organized, low-entropy state at the expense of free energy.

Cells cannot use heat for energy (cells are very heat-sensitive!).

Energy released in exergonicreactions used to drive endergonic reactions.

Require energy released in exergonicreactions (ATP) to be directly transferred to chemical-bond energy in the products of endergonicreactions.

Endergonic/exergonic refer to free energy changes (ΔG). Endothermic and exothermic refer to ΔH. For many chemical reactions, entropy contributions are relatively small, so chemists usually refer to ΔH.For many biological reactions, entropy contributions are significant, so biochemists usually talk about ΔG.

L28L28--55

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ATP – Major Energy CarrierFormation of ATP requires the input of a large amount of energy, stored in the bond energy by joining Pi to ADP. This energy released when ATP converted to ADP and Pi.

ATP is the universal energy carrier of the cell.

ΔH ~ 30 kJ/molΔH ~ 30 kJ/mol

+ H2O

L28L28--66

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Metabolism: GlycolysisA bag of sugar can be stored for years A bag of sugar can be stored for years

with little conversion to COwith little conversion to CO22 and Hand H22OO

HoweverHowever: this conversion is basic to life : this conversion is basic to life --> need to accelerate it!> need to accelerate it!

Mother Nature’s Solution:Mother Nature’s Solution:

Glycolysis – the break-down of glucose to pyruvate, catalyzed by enzymes

• Embden-Meyerhof Pathway:universal pathway - occurs in essentially all organisms

• overall net gain of 2 ATP

L28L28--77

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The 10 Steps of Glycolysis – I

L28L28--88

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The 10 Steps of Glycolysis - II

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The 10 Steps of Glycolysis - III

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Enzymes: Why?Living organisms must be able to carry out chemical reactions which are

thermodynamically highly unfavorable

– Break and form covalent bonds

– Move large structures

– Make complex three dimensional structures

– Regulate gene expression

They do so through enzyme catalysisenzyme catalysis.

Enzymes have immense importance in a wide variety of fields:

• Genetic diseases are frequently defects in enzymes or increased/decreased levels of enzymes

• Many modern drugs exert effects by interacting with enzymes

• Used in food processing and in chemical industry

• Enzyme inhibitors are a foundation of biological weapons (as well as of some of the counter-measures)

Enzymes have immense importance in a wide variety of fields:

• Genetic diseases are frequently defects in enzymes or increased/decreased levels of enzymes

• Many modern drugs exert effects by interacting with enzymes

• Used in food processing and in chemical industry

• Enzyme inhibitors are a foundation of biological weapons (as well as of some of the counter-measures)

L28L28--1111

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Enzymes, Proteins and Amino AcidsEnzymes are proteins that act as catalysts for biochemical reactEnzymes are proteins that act as catalysts for biochemical reactionsions.

Proteins are very large biomolecules present in living cells (~50% dry wt of our body).

All proteins are composed of the same building blocks - αα--amino acidsamino acids.

H

R

+H3N-C-C-O-

OH

R

H2N-C-C-OHO

or “zwitter-ion”(at neutral pH)

non-ionicform

H

R

H2N-C-C-OHO

+ H2OH

R’

H2N-C-C-OHO

H

H

R

H2N-C-C-N-C-C-OH

O OH

R’

amide group

+ =

α-amino acids are linked by amide groups, which are formed in a condensation reaction between the acid and amine groups of two amino acids:

This bond is also referred to as ‘peptide bond’, and the resulting molecules are ‘peptides’.

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Formation of a Peptide Bondgeneral reaction scheme:general reaction scheme: example:example:

Amino AcidAmino Acid PeptidePeptide Poly-PeptidePoly-Peptide Protein (e.g. Enzymes)Protein (e.g. Enzymes)

Peptides, Polypeptides, and Proteins (among them: Enzymes) are ‘Peptides, Polypeptides, and Proteins (among them: Enzymes) are ‘amino acid polymers’! amino acid polymers’!

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The 20 Amino Acids Found in Proteins

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Names & Types of EnzymesEnzyme names (mostly) end in Enzyme names (mostly) end in ––asease- identifies a reactant: sucrase - reacts sucrose, lipase - reacts lipid

- common names of digestion enzymes still use –in: pepsin, trypsin

- describes function of enzyme:

Class Reactions catalyzed

Oxidoreductases

Transferases

Hydrolases

Lyases

Isomerases

Ligases

ClassClass Reactions catalyzedReactions catalyzed

OxidoreductasesOxidoreductases

TransferasesTransferases

HydrolasesHydrolases

LyasesLyases

IsomerasesIsomerases

LigasesLigases

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Learning Check E1Match the type of reaction with the enzymes:

(1) aminase (2) dehydrogenase

(3) Isomerase (4) synthetase

( ) Converts a cis-fatty acid to trans.

( ) Removes 2 H atoms to form double bond

( ) Combine two molecules using ATP

( ) Adds NH3

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Enzyme Structure

The threeThe threecrucial for their functionality.crucial for their functionality.

Four hierarchical levels of enzyme structure are Four hierarchical levels of enzyme structure are distinguished: distinguished: primary, secondary, tertiary and quaternary.primary, secondary, tertiary and quaternary.

--dimensional structure of enzymes is dimensional structure of enzymes is

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Enzymes: Primary Structure

Primary structure: sequence of amino acids

glycine (G)

alanine (A)

valine (V) leucine (L)

serine (S) threonine (T)

cysteine

methionine (M)

phenylalanine (F) tyrosine (Y)

proline

isoleucine (I)

lysine (K)

tryptophan (W)

histidine (H)

asparticacid (D)

glutamicacid (E)

aspargine (N) glutamine (Q)

arginine (R)

3++2

: KVFGRCELAAAMKRHGLDNY…

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Primary Structure of Bovine InsulinFirst protein to be fully sequenced;Fred Sanger, 1953. For this, he won his first Nobel Prize (his second was for DNA sequencing).

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Enzymes: Secondary Structure

Alpha-Helix Beta-Sheet

Secondary structure: packing of amino acids (helix, sheet),i.e. the spatial arrangement of the ‘back-bone’ of the enzyme (without special consideration of side groups).

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Enzymes: Tertiary Structure

ribonuclease Aloop(non-repeating coil structure)

alpha-helix beta-sheetTertiary structure: cross-linking and 3D conformation,i.e. complete spatial arrangement of one enzyme

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Enzymes: Quaternary StructureQuaternary structure: enzyme oligomers, i.e. spatial arrangement of enzymes (and other peptides) which consist of several sub-units.

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Enzyme Structure: RecapFour hierarchical levels of enzyme structure:Four hierarchical levels of enzyme structure:

Primary structure:sequence of amino acids (1D)

Primary structure:sequence of amino acids (1D)

Secondary structure:spatial arrangement of backbone (2D)

Secondary structure:spatial arrangement of backbone (2D)

Tertiary structure:detailed spatial conformation of one enzyme (3D)

Tertiary structure:detailed spatial conformation of one enzyme (3D)

Quaternary structure:spatial conformation of multiple enzymes (‘oligomers’)

Quaternary structure:spatial conformation of multiple enzymes (‘oligomers’)

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Enzyme Action: Models

‘‘Lock and Key’ ModelLock 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

Induced Fit ModelInduced Fit Model

Enzyme structure flexible, not rigid

Enzyme and active site adjust shape to bind substrate

Increases range of substrate specificity

Shape changes also improve catalysis during reaction-> transition-state like configuration

In each case, an enzyme-substrate complex is formed, the respective bonds in the substrate are formed or broken

(i.e. the reaction occurs), and the product(s) are released:E + S <=> ES <=> E + P

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‘Lock and Key” Model

(A) The folding of the polypeptide chain typically creates a crevice or cavity on the proteinsurface. This crevice contains a set of amino acid side chains disposed in such a way thatthey can make noncovalent bonds only with certain ligands.

(B) Close-up view of an actual binding site showing the hydrogen bonds and ionic interactions formed between a protein and its ligand (in this example, cyclic AMP is the bound ligand).

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Induced Fit Model

Induced Conformational Change in Hexokinase

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Enzyme-Substrate Interaction