Waltho Flashcards
What are the diff classes of phosphate monoesters, and what are their roles?
- 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
What are the diff classes of phosphate diesters, and what are their roles?
- nucleotide transferases = transfer to co-substrate or enzyme
- nucleases = transfer to water
How does the structure of phosphate monoesters and diesters differ?
- DIAGS*
- diester has extra R group
Why does phosphate ester hydrolysis req catalysis?
- v slow reaction
What is the mechanism for chemical step for phosphoryl transfer w/o intermediates?
- DIAG*
- ld or la path
- both form same product
What is the energy profile for phosphoryl transfer?
- DIAG*
- higher energy before transfer
What enzymes can be involved in phosphate hydrolysis, and are they similar?
- 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
What are the pot pathways for phosphoryl transfer, what atoms are involved, what bond orders and distances?
- 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
What do diff values for bond order mean?
- 0 = no bonds
- 1 = single bond
- 2 = double bonds
What is the role of β-phosphoglucomutase (βPGM) and how is this achieved?
- 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
What is a phosphorane?
- pentavalent phosphorus
Why was it suspected the phosphorane in βPGM might actually be MgF3-?
- 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
Why was it disputed than MgF3- was in βPGM?
- unsure it existed as never observed in solution
- Mg strongly prefers to be 6-coordinate not 5-coordinate
What did synthesis of βPGM show?
- it synthesises a transition state analogue
- rather than stabilising (high energy) intermediate as would expect
What did studying validity of 2 models w/ NMR show?
- 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-
How can enzyme be trapped in diff parts of reaction cycle?
- can use diff species
- eg. Be to make BeF3 (tetrahedral) but doesn’t react as not Mg and Be v poisonous
How was a detailed pic of near transition state complex achieved?
- looked at e- distribution and proton distribution
How are charges balanced at expense of charge in βPGM?
- 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
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?
- 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
How can enzymes have a complex conformational landscape?
- multiple points can arrest, so can build up pic of entire catalytic process, inc substrate binding etc.
How do some near attack conformations mask nucleophile from target anion?
- 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
Why is βPGM reaction so slow when uncatalysed?
- water can’t get itself into correct orientation to do attacking
- gets into H-bonded position and can’t get past it
