protein structure, function and the enzymes of glycolysis triosephosphate isomerase

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

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.

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.

Proteins fold in such a way that they create specific sites that are the right size, shape, and polarity for their ligands.

Triosephosphate isomerase

Substrate =Dihydroxy acetonephosphate

Ligand binding is highly selective

The binding site is created by non covalent interactions

between the ligand and specific amino acid side

chains

Michaelis-MentonMichaelis-Mentonhyperbolic kinetics

The hyperbolic curve is defined by two parameters: Vmax and Km

Vmax

Km

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.

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.

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]

glucose glucose

pyruvate pyruvate

glycolysisgluconeogenesis

Many steps are shared. But, parallel pathways of catabolism and anabolism must differ in at least one step.

A

B

C

D E

F

The enzymes that catalyze the reactions that are different are targets for allosteric regulation.

Allosteric means “different site.”

Reactions that have a large G in either directionare generally different for the forward vs reversepathways.

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)

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)

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.

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.

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.

Feedback regulation:

End products of a metabolic pathway can act as allostericregulators of the initial steps of that pathway.

A B C D

phosphofructokinase

F6P + ATP ------> F1,6 BP + ADP

AMP +ATP -citrate -F2,6BP +

F1,6 bisphosphatase

F1,6 BP + H2O ----> F6P + Pi

AMP -F2,6BP -

How does allosteric regulation

work?

V

S

Sigmoidal Kinetics

V

S

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.

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.

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”

Proteins are:Function ExampleEnzymes DNA polymeraseStructural collagenTransporters hemoglobinMotors myosinStorage molecules caseinSignaling molecules insulinReceptor molecules rhodopsinRegulatory molecules lactose repressorSpeciality molecules antif reeze

Sizes and Shapes of Proteins

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

Side chains determine protein structure

Nonpolar amino acids form a hydrophobic core hidden from

water

Secondary structure is stabilized by hydrogen bonds.

Three types of noncovalent bonds stabilize protein folding.

The alpha-helix

Some proteins, such as cytochrome b are composed almost entirely of alpha-helices.

The beta-sheet

sheets can be parallel or antiparallel

The combination of helices and sheets constitute a protein’s secondary structure.

The enzyme phosphoglucomutase from the

glycolytic pathway.

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.

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

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

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.

Sizes of Proteins

Review

What types of noncovalent bonds help proteins fold?

Name a covalent bond that stabilizes a protein’s three dimensional structure.

The binding site is determined by amino acid

side chains

Antibodies selectively bind to antigens

How do enzymes catalyze reactions?

Lysozyme catalyzes the cutting of a polysaccharide

chain

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

Protein phosphorylation can increase or decrease enzyme

activity