Explain the two possible chemical routes for isomerization between aldoses and ketoses! Name one example each for an aldose-ketose isomerase using this mechanism! Name experimental strategies to distinguish between the two alternative mechanisms for a given isomerase! Which enzymatic reaction steps are particularly difficult to catalyze?
Enolization: phosphoglucose isomerase, triosephosphate isomerase
in tritium exchange experiments, T+ concentrations in the medium increase
Hydride shift: xylose isomerase
in tritium exchange experiments, T+ is not present in the medium
Difficult to catalyze in enolization: abstraction of H+ by glutamate (unlikely; has a much lower pKa than C-H)
Name three different enzymatic reaction mechanisms used by racemases! Name one example each and name commonalities and differences between the three mechanisms?
PLP-dependent racemases: alanine racemase
conversion of L-/D-amino acids
requires organic cofactor
forms Schiff-base
Two-base iso-mechanism: Proline racemase
conversion of D-/L-proline
quite similar to mandalate racemase but without metal ion
Metal-catalized enolization and two-base iso-mechanism: mandalate racemase
contains Mg2+ to lower the pKa of the mandalate alpha-atom
active site has lysine and histidine residues, one of which is always protonated to neutralize CO2-
assumption: Glu forms LBHB with mandalate to stabilize the enolate
Commonalities:
all catalize racemization reactions, most of amino acids
mandelate racemase and PLP work by stabilizing the negatively charged intermediate, but in different ways (mesomery vs. metal)
mandelate-DH and proline DH both use two-base iso-mechanism
Radical isomerases: Name the two radical initiators cofactors including the mechanism of radical formation! Name one enzyme for each cofactor!
General reaction:
Adenosylcobalamin (B12): glutamate mutase
radical formation
mechanism of the reaction (not important?)
S-adenosylmethionin (SAM) + Fe-S cluster: lysine 2,3-aminomutase
Describe briefly the mechanism of phosphomutases!
both (phosphorylated) OH groups are equally close to the catalytically active serine
both phosphates can potentially be abstracted by the serine
Explain two different mechanisms of epimerases acting on sugar substrates!
NAD+-based:
* deprotonated tyrosin (-O) is involved in forming the keto-function by abstracting the proton
substrate is bound via UDP to ensure free rotation —> non-stereospecific hydride transfer
Metal-based:
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