protein structure, function and the enzymes of glycolysis triosephosphate isomerase
TRANSCRIPT
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Protein Protein Structure, Structure, Function Function
and and The The
Enzymes of Enzymes of GlycolysisGlycolysis
Protein Protein Structure, Structure, Function Function
and and The The
Enzymes of Enzymes of GlycolysisGlycolysistriosephosphate isomerase
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How Proteins Work
How Proteins Work
Proteins recognize and bind to other molecules.
The bound molecule is called a ligand.The region of a protein that associates
with substrates and products is called the active site.
The region of a protein that associates with activator or inhibitor molecules is called an allosteric site.
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hexokinasecatalytic domain
This model was created in Chemscape Chime from the 2YHX pdb file by C.M.ANDERSON,R.E.STENKAMP,T.A.STEITZ.
Red indicates helix. Yellow indicates sheets. OTG (a glucose analog shown in white) is bound at the active site.
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Proteins fold in such a way that they create specific sites that are the right size, shape, and polarity for their ligands.
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Triosephosphate isomerase
Substrate =Dihydroxy acetonephosphate
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Ligand binding is highly selective
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The binding site is created by non covalent interactions
between the ligand and specific amino acid side
chains
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Michaelis-MentonMichaelis-Mentonhyperbolic kinetics
The hyperbolic curve is defined by two parameters: Vmax and Km
Vmax
Km
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Maximum velocity or Vmax is the maximum velocity of the reaction when the enzyme is saturated with substrate.
Turnover rate: the number of substrate molecules converted to product per second.
catalase has a turnover rate of 93,000.
DNA polymerase has a turnover rate of 15.
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Km is the substrate concentration at which the reaction velocity is equal to one half the maximal velocity (Vmax).
Values for (Km) are in the range of 10-1 to 10-7 M.
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Many reactions are shared. For these, G0 is usually either slightly positive or slightly negative. Thus, the direction of the reaction is dependent on the [reactant] and [product].
For example:
G6P F6P
G0 = + 1.7 kJ/mole; G = -2.5 kJ/mole
G= G0 + RT ln
[products][reactants]
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glucose glucose
pyruvate pyruvate
glycolysisgluconeogenesis
Many steps are shared. But, parallel pathways of catabolism and anabolism must differ in at least one step.
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A
B
C
D E
F
The enzymes that catalyze the reactions that are different are targets for allosteric regulation.
Allosteric means “different site.”
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Reactions that have a large G in either directionare generally different for the forward vs reversepathways.
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Glycolysis Reaction Step What is happening?*1) glucose + ATP --> G6P + ADP
(hexokinase)
2) G6P --> F6P(phosphoglucoisomerase)
*3) F6P + ATP --> F1,6 bisphosphate + ADP
(phosphofructokinase)
4) F1,6bisP ---> G3P + DHAP(aldolase)
5) DHAP--> G3P(triosephosphate isomerase)
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Glycolysis Reaction Step
What is happening?
6) G3P + NAD+ + Pi ---> 1,3 BPG + NADH(glyceraldehyde 3-phosphate dehydrogenase)
7) 1,3 BPG + ADP ---> 3 PG + ATP(phosphoglycerate kinase)
8) 3PG --> 2PG(phosphoglycerate mutase)
9) 2PG ----> PEP(enolase)
*10) PEP + ADP ---> pyruvate + ATP
(pyruvate kinase)
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Hexokinase has a regulatory domain as well as a catalytic domainTo see more, click on the
hexokinase pdb file link on the ISAT 350 home page.
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Hexokinase is inhibited by glucose 6 phosphate. Hexokinase is found throughout body.
By contrast, glucokinase is only found in liver and is not inhibited by G-6-P.
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Km is measured in concentration units. The higher Km, the weaker the substrate binds.
Typically, Km is close to the normal cellular concentration of the substrate.
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Feedback regulation:
End products of a metabolic pathway can act as allostericregulators of the initial steps of that pathway.
A B C D
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phosphofructokinase
F6P + ATP ------> F1,6 BP + ADP
AMP +ATP -citrate -F2,6BP +
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F1,6 bisphosphatase
F1,6 BP + H2O ----> F6P + Pi
AMP -F2,6BP -
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How does allosteric regulation
work?
V
S
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Sigmoidal Kinetics
V
S
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Proteins in RasMol and Chemscape Chime
Ribbon view of pyruvate kinase (catalyzes the last step in glycolysis)
In this view, the various colors correspond to individual subunit chains.
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Proteins in RasMol and Chemscape Chime
Spacefilling view of triosephosphate isomerase
In this view, red corresponds to regions with a helical structure, yellow regions are beta sheets
and white regions are randomly structured regions.
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Protein Structure Review:
http://www.massey.ac.nz/~wwbioch/Prot/tutehome/tutepage.htm
Go to the link at Massey University
Use their interactive tutorial on Protein Structure and Function to answer the question in
“questions.doc”
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Proteins are:Function ExampleEnzymes DNA polymeraseStructural collagenTransporters hemoglobinMotors myosinStorage molecules caseinSignaling molecules insulinReceptor molecules rhodopsinRegulatory molecules lactose repressorSpeciality molecules antif reeze
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Sizes and Shapes of Proteins
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Proteins are composed of amino acids.Amino acids are linked by peptide bonds to
form the primary structure of a protein.There are 20 different amino acids, each
with unique side chains.The sequence of amino acids and the
chemistry of the side chains determines how the protein folds which, in turn determines the protein structure and function.
How Proteins FoldHow Proteins Fold
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Side chains determine protein structure
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Nonpolar amino acids form a hydrophobic core hidden from
water
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Secondary structure is stabilized by hydrogen bonds.
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Three types of noncovalent bonds stabilize protein folding.
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The alpha-helix
Some proteins, such as cytochrome b are composed almost entirely of alpha-helices.
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The beta-sheet
sheets can be parallel or antiparallel
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The combination of helices and sheets constitute a protein’s secondary structure.
The enzyme phosphoglucomutase from the
glycolytic pathway.
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Proteins have several levels of organization
Proteins can form higher levels of organization such as the coiled-coil of two alpha-helices shown.
The three-dimensional conformation of a protein is referred to as the tertiary structure.
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SubunitsTwo or more polypeptide chains (subunits) can be joined to form a protein such as the CAP protein shown.
When a protein has more than one polypeptide, the complete structure is designated the quaternary structure
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Disulfide bonds can covalently join two parts of the same protein or two different poylpeptides
Disulfide bonds can covalently join two parts of the same protein or two different poylpeptides
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Protein Domains
Different parts of a polypeptide chain can fold independently to form a stable structure called a domain.
The different domains of a protein often have different functions such as the DNA binding domain (small) and the cyclic AMP binding domain of the CAP protein shown.
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Sizes of Proteins
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Review
What types of noncovalent bonds help proteins fold?
Name a covalent bond that stabilizes a protein’s three dimensional structure.
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The binding site is determined by amino acid
side chains
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Antibodies selectively bind to antigens
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How do enzymes catalyze reactions?
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Lysozyme catalyzes the cutting of a polysaccharide
chain
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The level and activities of an enzyme are regulated
Gene expression can be regulated by the amount of substrate (the lac operon)
Compartmentalization (proteases confined to the lysosome)
Changes in conformations (allosteric changes)
Protein phosphorylation
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Protein phosphorylation can increase or decrease enzyme
activity