Waltho

Question 1

What are the diff classes of phosphate monoesters, and what are their roles?

Answer 1

• kinases = put phosphate groups onto things
• phosphatases = remove phosphate group
• phosphomutases = rearrange position of phosphate (important in metabolism) w/in same substarte or to/from enzyme

Question 2

What are the diff classes of phosphate diesters, and what are their roles?

Answer 2

• nucleotide transferases = transfer to co-substrate or enzyme
• nucleases = transfer to water

Question 3

How does the structure of phosphate monoesters and diesters differ?

Answer 3

• DIAGS*

- diester has extra R group

Question 4

Why does phosphate ester hydrolysis req catalysis?

Answer 4

• v slow reaction

Question 5

What is the mechanism for chemical step for phosphoryl transfer w/o intermediates?

Answer 5

• DIAG*
• ld or la path
• both form same product

Question 6

What is the energy profile for phosphoryl transfer?

Answer 6

• DIAG*

- higher energy before transfer

Question 7

What enzymes can be involved in phosphate hydrolysis, and are they similar?

Answer 7

• kinases, phosphatases, phosphomutases, G-proteins
• not structurally related
• but lots in common at important parts of active site –> always O anion
• Mg metal of choice to neutralise phosphate
• mechanisms v similar

Question 8

What are the pot pathways for phosphoryl transfer, what atoms are involved, what bond orders and distances?

Answer 8

• metaphosphate
• -> axial O-P-O, bond order = 0, distance = 6Å
• concerted
• -> axial O-P-O, bond order >0 and <2
• phosphorane
• -> axial O-P-O, bond order = 2, distance = 3.5Å

Question 9

What do diff values for bond order mean?

Answer 9

• 0 = no bonds
• 1 = single bond
• 2 = double bonds

Question 10

What is the role of β-phosphoglucomutase (βPGM) and how is this achieved?

Answer 10

• interconverts glucose β-1-phosphate (G1P) and β-glucose 6-phosphate (G6P)
• essential aspartic acid phosphorylated in active enzyme and then phosphoryl group transfers to either 1’-OH of G6P or 6’-OH of G1P

Question 11

What is a phosphorane?

Answer 11

• pentavalent phosphorus

Question 12

Why was it suspected the phosphorane in βPGM might actually be MgF3-?

Answer 12

• energetics not predictable
• P-O bond lengths didn’t fit data
• X-ray measurements did not fit P in middle
• crystals were grown in 100nM NH4F, so F should cleave Asp-phosphate

Question 13

Why was it disputed than MgF3- was in βPGM?

Answer 13

• unsure it existed as never observed in solution

- Mg strongly prefers to be 6-coordinate not 5-coordinate

Question 14

What did synthesis of βPGM show?

Answer 14

• it synthesises a transition state analogue

- rather than stabilising (high energy) intermediate as would expect

Question 15

What did studying validity of 2 models w/ NMR show?

Answer 15

• 31P NMR showed no evidence for a phosphorane

- 19F NMR showed βPGM synthesises previously unknown species MgF3- in active site and MgF3- isoelectronic w/ PO3-

Question 16

How can enzyme be trapped in diff parts of reaction cycle?

Answer 16

• can use diff species

- eg. Be to make BeF3 (tetrahedral) but doesn’t react as not Mg and Be v poisonous

Question 17

How was a detailed pic of near transition state complex achieved?

Answer 17

• looked at e- distribution and proton distribution

Question 18

How are charges balanced at expense of charge in βPGM?

Answer 18

• enzymes make analogues with single -ve charge –> either AlF4- or MgF3- (NOT AlF3)
• up to pH 9.4 make AlF4- (octahedral)
• trying to mimic trigonal PO3- species
• so must be prioritising charge over correct geometry
• at high pHs make MgF3, which has right charge and geometry
• but AlF4- more stable
• transition state analogue complexes not affected by pH –> phosphate group isolated from effects of env

Question 19

What was the result of experiments which changed enzyme behaviour to see if inorganic chem or enzyme in charge, by seeing if inorganic chem reacts?

Answer 19

• looked at active site of βPGM in MgF3- and G6P
  
  • -> K145 sidechain and Fb replaced by water molecules in K145A variant
  • -> charge neutralisation occurs in spheres of radii >5Å from central Mg2+
  • -> so enzyme in charge
• ID 19F of βPGM TSA comlexes
• -> WT enzyme spopulate MgF3- and Al4- TSA complexes
• -> variant K145A populates MgF2 TSA complex
• NH4+ binding repopulates MgF3- TSA complex
• equivalent behaviour occurs in other phosphoryl transfer enzymes

Question 20

How can enzymes have a complex conformational landscape?

Answer 20

• multiple points can arrest, so can build up pic of entire catalytic process, inc substrate binding etc.

Question 21

How do some near attack conformations mask nucleophile from target anion?

Answer 21

• H bonded NAC decreases repulsion of incoming nucleophile through which H-bonding w/ phosphate surrogate
• aligned NAC has nucleophile in line for attack
• in presence of BeF3 and G6P, H-bonded NAC dominates in solution, although aligned NAC has measurable pop

Question 22

Why is βPGM reaction so slow when uncatalysed?

Answer 22

• water can’t get itself into correct orientation to do attacking
• gets into H-bonded position and can’t get past it