334296 enzymes and proteins

8/3/2019 334296 Enzymes and Proteins

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Introduction to Proteins andEnzymes

Basics of protein structure and composition The life of a protein

Enzymes Theory of enzyme function

Not all enzymes are proteins / not all proteins areenzymes

Enzyme REGULATION

Setting up an enzyme assay Buffer, cofactors, substrate, enzyme


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Why are Proteins Important?

Major class of catalysts in the cells;responsible for metabolism

Proteins interconvert energy

Proteins permit selective import and export ofmolecules from cells and organelles

Proteins allow movement

Proteins provide structural support for theorganism and individual cells


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What is a protein?

Linear chain of amino acids Amino acid: NH3 - CX - COOH

20 naturally occurring ones; essential

and nonessential Contains 3 or 4 hierarchies of structure

Primary structure: sequence

Secondary structure: alpha helix, beta strand, turn,[random coil]

Tertiary structure: beta barrel, ARM repeats, helix-

loop helix, etc. Sometimes contains quaternary structure (protein-

protein interactions)

QuickTime and aTIFF (Uncompressed) decompressor

are needed to see this picture.


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Properties of Amino Acids

http://dwb.unl.edu/Teacher/NSF/C10/C10Links/www.ccp14.ac.uk/ccp/web-mirrors/llnlrupp/Xray/tutorial/protein_structure.htm


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AminoAcid



Peptide BondFormation(a condensation

reaction)


More on Primary Structure


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Peptide BondFormation(a condensation

reaction)


are needed to see this picture.Peptide BondFormed



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, and Hydrogen Bonds,Dictate Secondary Structure



http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/protein.htm


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An Alpha Helix



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Three representations of analpha helix




are needed to see this picture.QuickTime and a

TIFF (Uncompressed) decompressorare needed to see this picture.

Ribbon, orLinguini diagram

Ball and stick Backbonehttp://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/protein.htm


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Beta strands are connected by hydrogen bonds

Alpha helix

QuickTime and a


Two beta strands



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Beta Strains Make PleatedSheets





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Helices and Strands CanMake Folds



EF hand in

Calmodulin(helix turn helix)

A SimpleExampleof TertiaryStructure



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Other Common Folds





http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/glycolysis.htm#tim

Beta barrelAB barrel


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Polypeptides Can Associate toForm Quaternary Structures

http://molvis.sdsc.edu/fgij/fg.htm?mol=2hhd

Hemaglobin

(deoxy form)


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You can observe proteinsdynamically in three

dimensions

http://molvis.sdsc.edu/fgij/index.htm

Jmol website


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The Life of a Protein

Synthesis Folding

Targeting

Function

Death


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Proteins are Polymerized onRibosomes



http://www.elmhurst.edu/~chm/vchembook/584proteinsyn.html


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Protein Elongation





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Protein Termination





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Protein Synthesis WebAnimation

http://www.johnkyrk.com/DNAtranslation.html


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Evolution of proteins Some pre-RNA polymer is believed to have

come first; RNA evolved from that

RNA can function as enzymes

RNA became the template for DNA Amino acids are attracted to codons

RNA catalyzed condensation of amino acids

Ribosomes evolved to increase efficiency andfidelity


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Folding of proteins Structure of proteins in inherent in primary

sequence (shown first for ribonuclease byAnfinson)

Proteins fold to reach their lowest energy

But cytoplasm is full of molecules; proteinswould self-fold with difficulty

Chaperones bind hydrophobic surfaces andprotect proteins during folding

Misfolding results in aggregation (Alzeimers

disease, prion disease)


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Folding Occurs to ReachLowest Energy

http://cnx.org/content/m11467/latest/




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Protein Targeting About half of proteins

are destined for otherlocations in the cell.

They have targetingsequences to getthem there.

Many accessoryproteins are required

for targeting.



http://www.windows.ucar.edu/earth/Life/cell_intro.html


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Mitochondrialimport, 2006QuickTime and a


Rehling and Pfanner (2004) Nat Rev MCB


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Protein Death

QuickTime and a


http://cellbio.utmb.edu/cellbio/lysosome.htm

Death in the Lysosome



http://en.wikipedia.org/wiki/Proteasome

Death in the Proteosome


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Enzymes

What is an En me?


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What is an Enzyme?

A protein molecule produced by living organismsthat catalyses chemical reactions of othersubstances without itself being destroyed or

altered upon completion of the reactions. Sometimes require cofactors (often metals or

vitamin derivatives)

Six types of reactions: oxidoreductases,transferases, hydrolases, lyases, isomerasesand ligases.

Ribozymes: RNA-based enzymes

http://cancerweb.ncl.ac.uk/cgi-bin/omd?query=enzyme&action=Search+OMD
http://cancerweb.ncl.ac.uk/cgi-bin/omd?proteinhttp://cancerweb.ncl.ac.uk/cgi-bin/omd?proteinhttp://cancerweb.ncl.ac.uk/cgi-bin/omd?moleculehttp://cancerweb.ncl.ac.uk/cgi-bin/omd?organismshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?catalyseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?chemical+reactionshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?substanceshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?beinghttp://cancerweb.ncl.ac.uk/cgi-bin/omd?reactionshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?oxidoreductaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?transferaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?hydrolaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?lyaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?isomeraseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?ligaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?ligaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?isomeraseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?lyaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?hydrolaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?transferaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?oxidoreductaseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?reactionshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?beinghttp://cancerweb.ncl.ac.uk/cgi-bin/omd?substanceshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?chemical+reactionshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?catalyseshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?organismshttp://cancerweb.ncl.ac.uk/cgi-bin/omd?moleculehttp://cancerweb.ncl.ac.uk/cgi-bin/omd?protein


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Consider a Phosphatase

Function: Desphosphorylation ofproteins and nucleotides

Reaction:

R-O-PO3 + H2O --> R-OH + PO4H


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J Biol Chem, Vol. 274, Issue 13, 8351-8354, March 26, 1999

COMMUNICATION

A Model of the Transition State in the Alkaline Phosphatase Reaction*Kathleen M. Holtz, Boguslaw Stec, and Evan R. Kantrowi

B t i l Alk li Ph h t


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Bacterial Alkaline Phosphatase

http://www.jbc.org/cgi/content/full/274/13/8351


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Why study enzyme kinetics?

Elucidation of enzyme mechanisms Dependence on cofactors, pH, etc.

Role of enzymes in pathways

Rate-limiting step?

Discovery of true substrates and

products Inhibition by drugs


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Use of a Model Substrate toStudy AP

Para-nitrophenyl phosphate (p

NPP) Hydrolyzed to para-nitrophenol

O2N OHO2N O PO3H2 + PO4H3

+H2O

Clear Yellow


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What we shall do?

Divide up into 7 groups

Each group will perform a differentreaction:

1) Does zincstimulate?

2) Is magnesiumrequired?

3) Does the reaction

stop completely onice?

4) What happens with nosubstrate?

5) What if you lower the pH?

6) What if you add phosphate?

7) THE GOLD STANDARD

334296 enzymes and proteins

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