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?

Solution

1)

Answer:

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

i.e. His297 MR catalyses the conversion of (R)-mandelate to (S)-mandelate. As per expectation, the mutant of MR in which His 297 has been converted to asparagine (H297N) does not show detectable racemase activity. However H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)benzoylformate at a rate equal to that measured for wild-type enzyme.

H297N catalyzes the stereospecific exchange of the -proton of (S)- but not (R)-mandelate with D₂O solvent. In the presence of D₂O, there is exchange of function between His297 MR and Lys166 MR.

H297N MR catalyses the conversion of (S)-mandelate to (R)-mandelate in presence of D₂O solvent.