Basic Enzymology

• Characteristics and capabilities of a bacterium– Genotype: Coded for in the DNA including plasmids– Genes code for proteins– Proteins are or are responsible for the traits of the cell– Examples:

• Sugars present on O-antigen of LPS• Whether the bacterium has a flagellum• Being able to use the sugar sucrose as a nutrient

– Mostly these proteins are enzymes• All enzymes are proteins (except for some RNAs)

1

Enzymes

• Have a distinct 3D shape essential for activity

• Are biological catalysts– Speed up the rate of a chemical reaction

• Lower activation energy by holding molecule in a favorable place

– Not consumed or permanently changed– Highly specific

• Will react some with similar molecules especially if their concentration is high.

2

3Enzyme function depends on shape

Substrates

Product

Enzyme brings substrates together in active site, increasing the rate at which they react.

http://www.columbia.edu/cu/biology/courses/c2005/images/substratesarelig.6.gif

Decrease in activation energy 4

http://upload.wikimedia.org/wikipedia/commons/thumb/e/e3/Activation2_updated.svg/300px-Activation2_updated.svg.png

Basic enzyme kinetics 5

http://dept.physics.upenn.edu/courses/gladney/mathphys/images/michaelis-menton.gifhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/L/Lineweaver_Burk.gif

Lineweaver-Burk

Inhibition

• Enzymes can be inhibited by disrupting their 3D shape– Adding high concentration of salt– Large changes in pH– Increasing temperature

• Disrupts ionic attractions, H-bonds, etc.

• Chemicals can bind permanently to enzyme– Changing shape– Binding to active site, preventing chemistry or substrate

access

• Competitive inhibition– Inhibitor resembles substrate, competes for active site

6

Competitive inhibition 7

http://www.elmhurst.edu/~chm/vchembook/images/573compinhibit.gif

8Allosteric sites

In allosteric site, inhibitor is not reacted, but causes a shape change in the protein. The substrate no longer fits in the active site, so it is not chemically changed either.

ghs.gresham.k12.or.us/.../ noncompetitive.htm

9More about Enzymes

• Sometimes an enzyme needs help– Protein alone = apoenzyme– Helper molecule: cofactor

• Could be inorganic like a metal ion (Fe+2)• Could be organic coenzyme (like CoA, NAD)

– Apoenzyme + cofactor = holoenzyme.– Cofactors have an effect on nutrition

• Bacteria have certain mineral requirements.• Vitamins are cofactors that are needed in the “diet”.

Metal cofactors 10

Mg+2 ATP

http://www.bmsc.washington.edu/WimHol/grafx%20research%20sum%20part%201/dengjmbfig4a.jpg

Organic cofactors 11

http://www.biochem.arizona.edu/classes/bioc462/462b/graphics/LDH-Lehn4un1417-p538.jpg http://biominer.bime.ntu.edu.tw/magiicpro/Examples/P10933/MaxSup_3.1qfy_S.png

Acetyl CoA

http://www.steve.gb.com/images/molecules/cofactors/acetyl_coenzyme_A.png

12Metabolic reactions require enzymes

• Reactions operate in pathways:

A B C D Where A-D are different molecules

Each step is catalyzed by a different enzyme.

Many, many catabolic and anabolic reactions can take place in a cell; each reaction requires a different enzyme.

Thus, there are many different enzymes present in a cell and a great many genes in the DNA coding for them all.

Which way? 13

Depends on which direction gives a decrease in the free energy of the products cf. the reactants: a negative ∆G.

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/images/pep.gif

There is a standard free energy change (both reactants and products at 1.0 M) that indicates which direction the reaction would go.

Concentration important! 14

In the example, if another reaction removed lactate, the concentration would be kept low, driving the reaction left to right.This also lowers ∆G, meaning reaction goes left to right.