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CBMS 223 Biochemistry: introduction & Amino acids Shoba Ranganathan Dept. of Chemistry and Biomolecular Sciences

CBMS 223 Biochemistry: introduction & Amino acids

Shoba Ranganathan

Dept. of Chemistry and Biomolecular Sciences Building F7B, Room 121 T: 02 9850 6262; E: shoba.ranganathan@mq.edu.au

Biochemistry

• Course website on Moodle (you need to login first):

ilearn.mq.edu.au – check regularly for announcements

Check your Macquarie email – we send you unit information

Check www.timetables.mq.edu.au – lecture room numbers may change from next week.

• Lecture 1:

Overview of course

Lab and tutorial classes

Assessment

Text books

Assumed knowledge Amino acids (also theory for Prac 1)

  pH and its importance in biological systems 2
 pH and its importance in biological systems
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Checking your Macquarie email

• We will contact you only via your MQ email:

this is the only email provided to us by eStudent

• If you do not see this regularly, please add your favourite email address here!

• This is specially important for any last minute changes of rooms, tutor/demonstrator info, exam date, etc.

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Lab format and expectations

• Essential items to bring to lab sessions

Lab coat and safety glasses

Please wear covered shoes (thongs/sandals: NO) Practical Notes & Laboratory Book

Buy from Co-Op Bookshop

This is also your Lab notebook for evaluation.

• Usually in two separate parts

so expect to be there the full 4 hours

• Read lab notes before the class – there will be prac quiz at each lab (2 marks/15 marks).

• Absence from lab must be made up during other sessions if available, or backed up by a medical certificate to avoid getting a zero mark.

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Lab format and expectations

• Lab results must be written up in the practical notes and lab. book BEFORE leaving the lab.

Record all results and paste all graphs/spectra/ printouts in the space(s) indicated.

• The finished account of ALL the experiments must be submitted to the lab, for grading as set out in the prac notes:

Pracs 1 & 2: 29 Mar, 30 Apr or 2 Apr after

T3/P3

Pracs 3-5: 31 May, 1 Jun or 4 Jun at the Prac test.

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Tutorial format and expectations

• Tutorials (2 hours) complement lectures and the practicals.

Mathematical problems and questions

how to complete your practicals will be covered during the tutorial class

Print the Tutorials notes from Moodle.

• Answer Part A before your scheduled tutorial session.

• You may have to

Work these out on the board or

Provide the answers or the steps.

Your tutor will discuss Part A and provide clues as to how to solve Part B questions, a few of which may be completed in the class.

Additional marks (1 mark per tutorial) will be given to students who have completed Part A problems and can explain the solutions to the class.

 These marks are in addition to your prac book and prac test marks. •
 These marks are in addition to your prac book and prac test marks.
• Absence from tutorial may must be made up during other sessions if
available (special consideration form), or backed up by a medical
6 certificate (no bonus mark applicable).

Course Assessment

Protein structure assignment (problem based questions) 5% Mid semester test (multiple choice questions) 15% Completed
Protein structure assignment (problem based
questions)
5%
Mid semester test (multiple choice questions)
15%
Completed Practical Notes & Laboratory Book
(3% x 5 pracs)
15%
Laboratory test (problems on practicals)
5%
Final exam
60%
Tutorials (bonus marks: 1% x 5 tutorials)
5%
Successful completion of each assessment
task is required for passing the unit.
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Key Dates

Week(s) Weightage Date(s) What is scheduled? 5 5% March 26 (Mon) Assignment due on Moodle
Week(s)
Weightage
Date(s)
What is scheduled?
5
5%
March 26 (Mon)
Assignment due on Moodle
5/6
6%
29 Mar (Thu),
Submit the practical notes and lab. book
30 Mar (Fri) or
to the lab. for grading P1 and P2 upto
2
Apr (Mon)
5.55 pm, after scheduled P3/T3
9
15%
May 8 (Tue)
Mid-semester test during the scheduled
lecture hour (C5C T1 + other rooms)
12/13
• 5%
May 31 (Thu),
• Practical test during lab session
1
Jun (Fri) or
• 9%
• Submit the practical notes and lab.
4
Jun (Mon)
book to the lab. for grading P3-P5
Sem 1
60%
June 12
onwards
Exams
CBMS223 Exam (usually early!)
(check exam timetables by mid-April)
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Textbooks

BIOCHEMISTRY 4th Edition: Garret and Grisham:

Thomson Learning 2009 (recommended)

• BIOCHEMISTRY 3rd Edition: Garret and Grisham:

Thomson Learning 2007 BIOCHEMISTRY 3 rd or 4 th Edition: Voet and Voet: Wiley Publishing 2003/2004

• BIOCHEMISTRY 4th Edition: Zubay: WC Brown Publishing 1997 (ONLY available second hand)

• BIOCHEMISTRY Explained 1st Edition: Thomas Millar : Wiley Publishing 1999 (remedial text)

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Textbook for PG students lacking Organic Chemistry background

Organic Chemistry 9th Edition: T.W. Graham Solomons & Craig B. Fryhle, Wiley, 2009 (recommended)

Tips on doing well

Workload in the unit is fairly heavy. You’ll need to work steadily, because new material is based on earlier material. You will not be able to leave mastering the content until the last few weeks.

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Assumed Knowledge

• Chemistry

mole concept

carbon chemistry

chirality - mirror image compounds

pH and pKa

properties of functional groups

chemical equilibrium

elements of thermodynamics

• Biology

difference between a prokaryote and a eukaryote
ultrastructure i.e. organelles, etc. DNA to RNA to protein

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Mole concept

• 1 mole of a compound is a reference to the number of molecules of that substance i.e. 6.023 x 10 23 molecules (Avogadro’s number)

• This number of molecules of a compound is equal to the molecular weight of that compound in grams.

Moles (n) = weight (wt) / molecular weight (Mwt)

• The number of moles, ‘n’ of a substance in solution, equals the concentration of that substance, ‘c’ in moles per litre, multiplied by the volume in litres, ‘v’. n = cv

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Chirality or handedness

• Chiral compounds: non-superimposable mirror image forms also known as enantiomers. This property is characterised by optical activity.

most biological molecules have mirror image forms and only one of these forms usually occurs in nature.

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Course format

• Weeks 1-4

Properties of amino acids and proteins

importance of these properties in terms of protein structure (assignment)

proteins as catalysts: enzymes

• Weeks 4-9

Metabolic pathways

energy metabolism
anabolic and catabolic pathways
compartmentalization of these pathways
metabolism of sugars (upto here for mid-sem test) making ATP: electron transport and oxidative phosphorylation

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Course format

• Weeks 9-13

Metabolism of other key biomolecules

Nitrogen and amino acid metabolism Nucleotide metabolism Fats and fatty acid metabolism Vitamins and coenzymes

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Water: the medium of life

• Water is essential for all forms of life.

• Every organism is 70-90% water!

• Normal metabolic activity requires min. 65% water!

• Water is an excellent solvent.

• Water causes ionization of polar molecules, critical for the function of:

amino acids and proteins nucleotides and nucleic acids even phospholipids and membranes.

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Amino acid structure

• Amino acids are important because they are the building blocks of proteins

amino group and an acidic

• They

both

an

carboxyl group

• All amino acids have the general structure (in solution near neutral pH)

NH 3 + (CHR) CO 2 called a zwitterion: carrying both a

-

• This is

positive and a negative charge at the same time. 17
positive and a negative charge at the same time.
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Amino acid structure

Amino acid structure Solid or gas phase In aqueous solution at pH 7 18

Solid or gas phase

Amino acid structure Solid or gas phase In aqueous solution at pH 7 18

In aqueous solution at pH 7

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Amino acids found in proteins

• 20 common amino acids with different ‘R’ groups are found in proteins.

• Four main classes with different types of R groups have been identified:

Non-polar amino acids (8)

Polar, uncharged amino acids (7)

Acidic amino acids (2)

Basic amino acids (3)

• Each amino acid has a one letter code and a three letter code that are helpful when working with proteins that can contain hundreds of amino acids.

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Class I amino acids: Nonpolar (hydrophobic) - I

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Class I amino acids: Nonpolar (hydrophobic) - II

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Class II amino acids: Polar, neutral - I

Class II amino acids: Polar, neutral - I 22
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Class II amino acids: Polar, neutral - II

Class II amino acids: Polar, neutral - II 23
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Class III amino acids: Acidic

Class III amino acids: Acidic In solution, these amino acids are known as by the names

In solution, these amino acids are known as by the names of their ionic forms: aspartate and glutamate, respectively.

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Class IV amino acids: Basic

Class IV amino acids: Basic 25
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Uncommon amino acids occur in proteins

• Hydroxylysine, hydroxyproline - collagen

• Carboxyglutamate - blood-clotting proteins

• Pyroglutamate – in bacteriorhodopsin

• Phosphorylated amino acids – a signaling device

• Aminoadipic acid is found in proteins isolated from corn

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Structures of some rare amino acids

Collagen corn Bacterio- Blood-clotting 27 rhodopsin proteins
Collagen
corn
Bacterio-
Blood-clotting
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rhodopsin
proteins

Acid-base properties of amino acids pH: a measure of the acidity of a solution

H 2 O

• pH = -log 10 [H + ]

• for pure water, [H + ] and [OH - ] equal 10 -7 M

pure water, [H + ] and [OH - ] equal 10 - 7 M H +

H +

+

OH -

For pure water, [H + ] = [OH - ] = 1 x 10 -7 M & pH = 7

Importance of pH

• pH determines the viability of biochemical reactions

• It affects ionization and hence the charge of molecules that have acidic or basic groups (groups than can gain or lose protons).

This can have significant effects on the biological, chemical and physical properties of these molecules.

 • pH can thus have a significant effect on the properties of amino acids
• pH can thus have a significant effect on the properties of amino
acids and hence proteins.
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Amino acids are weak acids/bases - dissociation based on pH of media

• The most common equilibrium that biochemists encounter is that of weak acids and bases, both of which can affect the pH

HA

of weak acids and bases, both of which can affect the pH HA H + +

H +

+

A -

• Strong acids dissociate completely in solution but weak acids only partially dissociate and the equilibrium constant for this reaction is called the K a K a = [H + ] [A - ]

[HA] 29
[HA]
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pH of amino acids (weak acids/bases) from Henderson-Hasselbalch equation

• Taking logarithms of this equation gives us -log [H + ] = -log K a + log ([A - ]/[HA]) or pH = pK a + log (base/acid)

• When there is an equal amount of base and acid then pH = pK a

also known as half-titration point titration is an effective way to determine the pK a values

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pI

• pI is defined as the pH at which there is no NET charge on the amino acid (also for a protein or peptide).

• For simple systems such as amino acids and small peptides, it can be calculated easily.

• For more complex systems such as proteins, it can be measured experimentally.

• If the molecular structure is known, pI can be measured by

completely protonating the molecule,
titrating the number of positive charge equivalents and then measuring the pH.

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Most amino acids have two ionizable groups!

Acidic

Neutral

Most amino acids have two ionizable groups! Acidic Neutral pI Basic 1. 2. 32

pI

Basic

1. 2. 32
1.
2.
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Following the ionization of an amino acid:

1. Acidic to neutral pH

1.
1.

Start: low pH

• COOH group uncharged (weak acid!)

• Amino group is protonated: +1 charge With increasing pH (i.e. less acidic or more basic solution):

• COOH starts to lose its proton

• Equilibrium is getting established

As pH increases :

As pH

increases:

• Zwitterion predominates At equilibrium • Cationic and zwitterionic forms have equal concentration 33 •
• Zwitterion predominates
At equilibrium
• Cationic and zwitterionic forms have equal concentration
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• Equilibrium constant is called K 1 : in pH scale, this is pK 1
• Charge is +0.5 (average of the charges of the two ionic species)

Following the ionization of an amino acid:

2. Neutral pH to basic pH

2.
2.

From: neutral pH

• Both groups are charged

• Molecule is essentially neutral

• pH at zero charge = pI

With increasing pH (i.e. less acidic or more basic solution):

• Amino group starts losing its proton and thus, its charge

A second equilibrium is getting established As pH increases:

equilibrium is getting established As pH increases : • • Negative charge predominates At equilibrium •
• Negative charge predominates At equilibrium • Zwitterionic and anionic forms have equal concentration 34
• Negative charge predominates
At equilibrium
• Zwitterionic and anionic forms have equal concentration
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• Equilibrium constant is called K 2 : in pH scale, this is pK 2
• Charge is -0.5 (average of the charges of the two ionic species)

Ionic forms of an amino acid as pH changes

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pK values of amino acids are unique and can identify them!

• Backbone or -COOH group (pK 1 ) = ~2

• Backbone or -NH 2 group (pK 2 ) = ~9

• pI is also unique for each amino acid

The pI of a protein, which is made up of amino acids is thus dependent on its amino acid composition!

pI is used to separate different proteins

• Some amino acids also have additional ionizable groups in their sidechains

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p K values 37
p K
values
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So why the pK value of R group?

• Two amino acids have extra COOH groups in their sidechains (i.e. R groups)

The extra COOH is also a weak acid and can lose its proton!

The extra COOH is also a weak acid and can lose its proton! • Aspartic Acid,

• Aspartic Acid, Asp, D: pK R = 3.9

• Glutamic Acid, Glu, E: pK R = 4.3 38
• Glutamic Acid, Glu, E: pK R = 4.3
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More complications: sidechains with amino groups!

More complications: sidechains with amino groups! • Histidine, His, H: pK a = 6.0 • Lysine,
• Histidine, His, H: pK a = 6.0 • Lysine, Lys, K: pK a =
• Histidine, His, H: pK a = 6.0
• Lysine, Lys, K: pK a = 10.5
• Arginine, Arg, R: pK R
(guanidino group) = 12.5
So, which amino acid is the
strongest base?
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Summary

• Revision

Mole concept

n = wt / Mwt n = c v

Chiral molecules: non-superimposable mirror image forms of compounds (“handedness”)

• Amino acids

Acid-base properties of amino acids

Henderson-Hasselbalch equation Introduction to pH and pI pK of all amino acids pK R for some!

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