a 10 pts When His297 of mandalate racemase is mutated to Asn

a) [10 pts] When His297 of mandalate racemase is mutated to Asn, there is no detectable racemase activity. If we mix mutant enzyme and substrate together in D2O, it is possible to observe a chemical transformation with one of the mandalate enantiomers. Which enantiomer reacts, and what chemical reaction takes place (show starting material and product)?

b) [10 pts] Show an arrow pushing mechanism for the reaction that occurs. Show any enzymatic general acid/base residues (including Glu317), but you can omit other enzyme functional groups. Assume that any protons on general acid/base residues and hydroxyl groups have exchanged with deuterium.

c) [10 pts] Sketch a free energy profile for the mandalate racemase reaction following the profile we drew in class. You do not need to show the non-enzymatic reaction. Now sketch what the free energy profile looks like (qualitatively) for the His297Asn mutant of mandalate racemase. For simplicity, assume that the mutation only affects transition state stabilization (i.e. barrier heights), and that binding to substrates and/or intermediates is not affected.

d) [5 pts] If you run a reaction with wild-type mandalate racemase and R-mandelate in D2O, what product(s) will you observe when the reaction goes to completion?

e) [10 pts] If you run a reaction with wild-type mandalate racemase and S-mandalate in D2O, at very early timepoints (<5% conversion) you will observe deuterium incorporation into the Smandalate starting material at concentrations comparable to the amount of deuterated Rmandalate product formed. If you run a similar reaction starting from R-mandalate, at 5% conversion you will observe no significant deuterium incorporation into the R-mandalate starting material. What is happening?

I need help with a) though could also use help for parts d) and e) as well.

Solution

Mandelate Racemase is a bacterial enzyme, obtained from the soil bacteria Pseudomonas putida, catalyzes the interconversion of the enantiomers of mandelic acid.The mandelate racemase enzyme contains two general acid/base catalysts on its active site: Lys 166 and His 297. Lys 166 removes the alpha-proton from (S)-mandelate, and His 297 removes the alpha-proton from (R)-mandelate.

When His 297 is mutated to Asn by site-directed mutagenesis into H297N, there is no structural change. The mutation of His 297 inhibits the activity of mandelate racemase enzyme but does not affect the function of Lys 166 as a catalyst.

The H297N racemase turns inactive due to racemization but catalyses the exchange of deuterium from D₂O (alpha proton) of L-mandelate but not of D-mandelate. It can also remove bromide from L-P-(bromomethyl) mandelate but not from D-P-(bromomethyl) mandelate.

His 297 is bound to Asp 270 with a hydrogen bond and its mutation to Asn 270 reduces the activity of the mandelate racemase enzyme to around 10,000 times lesser than the wild type of enzyme.