Solute transport and the bacterial PTS

Question 1

What is TMG?

Answer 1

A galactoside analogue

Question 2

What are the two classes of E.coli lactose negative mutants?

Answer 2

Normal TMG uptake but lacking ß-galactosidase
Normal ß-galactosidase but deficient in TMG uptake
Therefore, both activities controlled by a gene

Question 3

What protein is TMG accumulation catalysed by?

Answer 3

Lactose permease by lacY from lac operon

Question 4

What are proteins involved in solute transported referred to as?

Answer 4

Permeases/carriers/transporters/transport systems

Question 5

Draw out bacterial transport systems

Answer 5

DO IT

Question 6

Draw basic PTS system (and with optional domain)

Answer 6

DO IT

Question 7

What are the properties of each of the PTS proteins?

Answer 7

Enzyme I and HPr are general PTS proteins, are involved in all the phosphotransferases in the cell (with a few exceptions); phosphorylated on a histidine
Enzyme II = substrate-specific, contains a number of domains, may be a single polypeptide chain or separate proteins
EIIA = contains first phosphorylation site, on a his residue
EIIB = contains second phosphorylation site, on a his or cys residue, depending on the system
IIC (and IID) = are not phosphorylated, are membrane-associated, involved in translocation

Question 8

Draw the glucose PTS for E. coli

Answer 8

DO IT

Question 9

Draw the mannitol PTS for E. coli

Answer 9

DO IT

Question 10

What is the functional relationship between PTSs?

Answer 10

PTS proteins from different organisms interact functionally to phosphorylate sugars

Question 11

What is the structural relationship between PTSs?

Answer 11

Sequence analysis shows homology between systems, however, during evolution various hydrophilic and hydrophobic domains have become fused in different orders and combinations, or spliced to become distinct polypeptide chains

Question 12

What are the standard free energies of hydrolysis between each stage of PTS, and what does that imply?

Answer 12

PEP -14.6kcal/mol
P~EnzI -14.5kcal/mol
P~HPr -13kcal/mol
P~IIA glc -14.4kcal/mol
Glucose-6-P -3.3kcal/mol
So phosphotransfer reactions up to IIA (and presumably IIB) are in equilibrium and the phosphate can be transferred in both directions