Horizons of biochemical research

- Aspartate side chain on the amino acid removes Cl- forming a covalent complex with the substrate

- This inverts stereochemistry and produces an ester 

- Enzyme activates H20

- H20 used to hydrolyse ester intermediate and form the product 

- Iodine is essential for all organisms 

- Bromine is used by marine organisms 

- Good H-bond acceptors, but do not make H-bonds 

- Good leaving groups 

- Broadly hydrophobic and improve the performance of a synthetic compound 

- Organisms use it to detoxify foods, if they can do this, removes competition 

- Enzymes that do this could help in complex chemical synthesis by allowing the reaction to happen at room temperature and specificity. 

- X- ray (crystalography): 

- Precisely visualizes enzyme and the distances between parts of the protein 

- NMR: 

- Precisely tells us what bonds are present but only good for small proteins

- EM: 

- Little sample required but ideal for studying larger proteins 

- Enzymes (20-60 kd) are usually too big for NMR but too small for EM - X-ray is best. 

- Both apo and intermediate structures seen 

- Mutant disabled in step 2 used to see intermediate complex

-Observations: 

- Intermediate pulls arginine towards the active site, stabilizing the removal of Cl- (may not see the importance of the arginine if we only had apo) 

- Arginine abstracts Cl-- Specific aspartate carries out the reaction 

- Specific aspartate carries out the reaction 

- Only a snapshot, cannot see dynamics, could be different? 

- Change environment to find out more information 

- Design of assay is critical 

- Cheap, fast, easy 

- If reaction needs to be stopped to measure product formation then the reaction could have already started stopping due to substrate running out

- Lots of mutants created

FINDINGS: 

- Histidine (H19) not essential 

- Tryptophan (W179) not essential (not Cl- acceptor) 

- Tyrosine (Y12) & tryptophan (W179) mutants less active (pocket for substrate) 

- Serine (S175) & Threonine (T14) needed for highest activity 

ESSENTIAL: 

- Arginine (R41) 

- Lysine (K151) 

- Asparagine (N177) 

CRITICISM 

- No error bars, no repeats 

- Picked up something working at 0.03%, good assay 

- Used to work out RLS 

- MS used to identify the aspartate 

- Inhibitor added

- If inhibition is fast, first step is RLS OR final step is RLS (active site empty) 

- If inhibition is slow, second step RLS (competition with substrate) 

- Change isotope of atom in reaction

- If the heavier atom is used in the RLS then the reaction will be slower

- If not in RLS rate will be unchanged 

- KIE: heavy atom/ light atom                        Larger atoms, larger ratio = worse 

- Primary KIE = Isotope is in the bond being made/broken in the RLS 

- Secondary KIE = Isotope is close to the bond making/breaking in RLS 

- Solvent KIE = Isotope part of the solvent (H/D) involved in RLS 

- KIE dependent on mass difference 

- 37/35 = 1.06 

- Chloride abstraction? mean ratio for both weights = 1.01 

- not rate limiting 

- Changing H for D in the water, observe O-H bonding breaking 

- S-enantiomer KIE = 1.48 +/- 0.1 = RL but not all of RLS 

- R-enantiomer KIE = 0.87 +/- 0.27 = not RL, water breaking no-longer RL in this enantiomer 

- Use isotoped to see which ends in the product 

- Allows us to see where the product comes from & tells us about mechanism 

Mechanisms: 

1. Enzyme side chain attacks haloacid, Cl- is lost, water is used to break the ester to form the product, oxygen from side chain 

2. Enzyme strips H from H2O, to form an attacking group that knocks off the halogen and form the product, oxygen from water 

- Set up reaction so that turns over once 

- Easier and cheaper to make the water heavy

- Measure MS

- Results = majority light, using heavy water, mechanism 1 correct.