Edition
Charlotte 1
Essential
Biochemistry
Second W. Pratt | Kathleen Cornely
Lecture 6: Feb 2, 2012
Chapter 5
Protein Function
Sections 5.2 thru 5.3
Copyright 2011 by John Wiley & Sons, Inc.
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Reminders
Homework for Chapters 4 and 5 are due midnight
Friday Feb 10
Exam 1 is scheduled for Tuesday 2-7
Chapters 1, 2, 4 and 5 are covered on the exam
It will be all selection-type questions (matching or
multiple choice)
It will be held at 7-9 pm
There will be assigned seating check Blackboard for
assignment
There is no lecture on Tuesday
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Amino Acid Game
Lets play the game:
http://www.wiley.com/college/boyer/0470003790/animations/anim
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Structural Proteins
Cytoskeleton proteins
Distributed differently in cell
Microfilaments (~70)
Actin
Intermediate filaments (~100)
Keratin
Collagen
Microtubules (~240)
Tubulin
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Actin Microfilament
ATP
G-actin
Two forms
Globular protein with ATP binding site: G-actin
Monomeric
Filamentous actin: F-actin
Double chain of subunits
(-) end has ATP site
(-)
(+) at opposite end
Monomers added together to form filament
Addition more rapid at (+) end
Driven by ATP hydrolysis
Catalyzed by F-actin
F-actin
Most recently added actin bound to ATP
(+)
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ATP Hydrolysis
Major way the cell has to release energy
stored in chemical bond
Input energy to form bond
Releases energy when broken
Phosphate is a high energy bond
Takes a lot of energy to hold 2 phosphates
together repulsion from like (-) charges
Breaking that bond releases a lot of energy
Major energy currency in cell
Can spend it in many ways
Diffusible send it where you need to
spend it
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Actin Dynamics
Polymerization of actin is reversible
Grows and shrinks over time
Treadmilling: when addition = subtraction of subunits
Assembly and disassembly regulated
Can be used to extend and retract cellular processes
One method of cellular motility
http://www.youtube.com/watch?v
=PsYpngBG394
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Microtubules
Built from dimers of tubulin
Forms hollow cylinder, rather than single
filament
More rigid
GTP
Think about bicycle frames
Forms protofilament (like actin)
Formation of microtubules
,tubulin
Each monomer binds GTP nucleotide
GTP blocked in but solvent-exposed in
Formation of microtubule causes hydrolysis of GTP
in but not in
Tubulin dimer
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Microtubules
Protofilaments (13) line up side by side to form tube
Dimers can add at both ends
(+) side faster
Disassembly faster at (+) end also
Used to construct cilia and flagella
Used to align and separate pairs of chromosomes during
mitosis
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Intermediate Filaments
Exclusively structural proteins
Keratin
Basic unit is dimer of coiled helices (coiled coil)
7-residue repeat: #1 & #4 residues nonpolar
Line up along one side of helix
Hydrophobic van der Waals interactions hold them
together
Dimers associate to form tetramers, etc.
Like winding several strands together to form strong rope
Part of hair
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Collagen
Holds cells together (French: glue)
Supports bodys weight
Trimeric molecule
Part of animal bones and tendons
Every third residue is Gly
30% of rest is Pro and Hyp (hydroxyproline)
Triplets of Gly-Pro-Hyp
Forms narrow, left-handed helix
Polypeptides wind around each other to
form right-handed triple helix
+ Collagen
Gly residues in center of triple helix
No other residue would fit!
Triple helix stabilized by H-bonds
After secreted from cell that synthesized
them, proteases trim them
Align side-to-side and end-to-end to form
large fibers
Fibers have strong cross-links through
oxidation of Lys chains
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Motor Proteins
Myosin and Kinesin
Mode of action different
Basic structure the same
Different functions in the cell
Myosin
2 polypeptide chains that form 2 heads
and a long tail
Head has binding site for actin and for
ATP
2 small polypeptides (light chains)
wound around the neck the of protein
2 heads act independently
Light
chain
Myosin
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Myosin Mechanism
Myosin tails associate to form thick
filament
Heads sticking out
Heads (multiple) bind to actin thin
filaments
Sequence of movements
1.
2.
3.
4.
Head bound to actin
Head binds ATP and releases actin
Hydrolysis of ATP rotates head
Head binds to actin subunit further along
This releases Pi and ADP
Release causes head to ratchet back
Power stroke
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Kinesin
Microtubule-associated motor protein
Similar structure to myosin
Shorter neck
Light chains at other end of polypeptides
Light chains bind some vesicle, with purpose of moving
it along a microtubule
Moves by stepping instead of grabbing
ATP hydrolysis drives the lever
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Mechanism of Kinesin
Leading head bound to subunit of protofilament
Leading head binds ATP and neck retracts
This swings the trailing head forward by 180 toward
the (+) end
Force-generating step
New leading head binds tubulin and releases ADP
Trailing head has bound ADP
This moves cargo forward
ATP hydrolyzed in new trailing head (ADP bound)
Trailing head detaches
Ready to start over
This is processive
One head always bound
Cargo remains attached and keeps moving along
http://www.youtube.com/watch?v=YAva4g3Pk6k&NR=1
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Essential
Biochemistry
Second Edition
Charlotte W. Pratt | Kathleen Cornely
Chapter 6
How Enzymes work
Section 6.1
Copyright 2011 by John Wiley & Sons, Inc.
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Need for Biological Catalysts
Half-live of peptide bonds in vitro is ~20 years
Only of peptide bonds hydrolyzed by water after 20
years!
Clearlya very stable bond, but
What if we want to recycle? How can we make it happen?
T: living organisms; can only tolerate so much
[reactants]: may be scarce inside cell
Add a catalyst
Speeds up reaction but not consumed in process
+ Enzymes as Catalysts
Enzyme from Grk in yeast
First used with reference to fermentation in yeast
1st recognition that component separable from living cell
Biological catalysts
Not permanently altered or consumed
Most are protein, though some are made of RNA
Can speed up reactions to an amazing degree
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+ Enzymes as Catalysts
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Each enzyme has an active site where catalysis takes place
Most often in crevices on the enzyme surface
Sometimes at subunit interface
Sometimes in pocket that closes around the substrate (protected)
Hexokinase binding glucose
Chymotrypsin
Active site residues in red
+ Enzymes as Catalysts
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Differ from metal catalysts:
Have complex, specific structures
Catalysis may be associated with conformational changes
Work under mild conditions (T and P)
Act only on a specific substance (substrate)
Some will act on the L-enantiomer, but not the D
Some are more promiscuous than others
This may be important for detecting and measuring enzyme
activity
Hydrolysis of p-nitrophenylacetate
can be followed by A410
Absorbs at 410 nm
+ Classes of Enzymes
Enzymes often regulated by a number of mechanisms
Knowing when and why they work is as important as how
Classes of enzymes
Addition or removal of one substance to another
Oxidoreductases
Transferases
Ligases
Hydrolases
Lyases
Rearrangements of substance
Isomerases
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+ Naming Enzymes
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Naming enzymes
Often named for substrate it acts on
Name most often ends in -ase
Name often tells you what it does
May be >1: multiple enzymes catalyzing same reaction
Isozymes (in different tissues or expressed under different
conditions
Often differ in catalytic properties
Transfers amine from alanine
Removes carboxyl from pyruvate
What class does the following
enzyme belong to?
A.
Oxidoreductases
B.
Transferases
C.
Ligases
D.
Lyases
E.
Hydrolase
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iClicker Question
How I feel about the exam next week:
A. No sweat I got this!
B. So far so good but Im not really
sure.
C. Im worried but Im going to study
this weekend and then Ill be OK.
D. OK but Im really worried about
that acid-base chemistry.
E. Im so lost!