1st phosphorylation of glucose by HK

Question 1

What and where does HK phosphorylate?

Answer 1

• any 6C sugar but primarily glucose.
• phosphoylates the a or b anomer due to mutarotation, can either do it at active site or. before the active site.
• SN2 LIKE mechanism used to transfer the phosphate (involves TS and is concerted and inverted prod)
• ## ONE Mg2+ is involved in this process, as it shields some of ATP’s negative charge so this is ELECTROSTATIC catalysis

Question 2

Mg and Se

Answer 2

• considered to be metals/semimetals
• found in active sites NOT as metalloenzymes (those are transition metals)

Question 3

HK has what kind of mechanism

Answer 3

random Bi Bi mechanism
- either will bind first and there are 2 substrates at the beginning and 2 products at the end

Question 4

what amino acid is in active site of HK

Answer 4

aspartate

Question 5

What is Aspartate doing in the active site specifically?

Answer 5

• 1. aspartate is going to be deprotonating the hydroxyl group at C6 of gluc (reason: SO THAT CAN BE A NUCLEOPHILE TO ATTACK ATP)
     (aspartate acting as a base, removes H)
• 2. also going to be involved in hydrogen bonding with C4 of gluc to set up proxoimity and orientation in order to do acid base catalysis

Question 6

HK catalyzes glucose P+ fastter than

Answer 6

ATP hydrolysis…
WHy?
active site has characteristics of an organic solvent… isolated water from active site.

Question 7

HK structure

Answer 7

• monomers have 2 domains with a hinge that moves up to 8 A
• this hinge movement to closed conf puts the Mg2+-ATP near the CH2-OH at carbon 6. (proximity and orientation)

Question 8

How is the closed conf stabilized?

Answer 8

The closed con is stabilized by Gluc+ATP binding, this stabilization will cause a conf selection then population shift, which is a shift in the equilibrium to favor the closed conformation.
- closed conformation is when the enzyme is doing its job, wont be stabilized if water is in the active site instead of glucose
- have now created a population that consists of more stabilized conformations, which allows you to do your reaction

Question 9

Population shifts are models of?

Answer 9

• enzyme regulation
• enzyme actually doing its job

Question 10

closed conformation

Answer 10

• active site amino acids from both domains are in close proximity
• closed active site enables greater nucleophilicity in reaction
  why? no water in active site, active sites approximating organic solvents, nucleophilicity better because the nucleophile is no longer shielded by water molecules