Explain possible chemical routes (4) to afford a decarboxylation of metabolites.
requires cofactor (X) as electron sink (e.g. PLP)
common mechanism
phosphate is a good leaving group and “makes” CO2 leave
relatively rare
example: mevalonate pyrophosphate decarboxylase
requires electron acceptor
example: formate-DH
radical must be generated before and quenched afterwards
Explain the mechanism of pyruvoyl-dependent decarboxylations!
Which reaction is catalyzed by acetoacetate decarboxylase? Draw the mechanism and explain the different functional roles of the two neighboring lysine residues!
Acetoacetate decarboxylase:
E-Lys115 has a particularly low pKa because of the hydrophobic environment —> NH2 (important for nucleophilic attack to form a Schiff-base)
Lys116+: necessary to keep Lys115 in hydrophobic pocket. (?)
Compare thiamine diphosphate-dependent and PLP-dependent decarboxylases! Name commonalities and differences in the mechanisms! Describe how PLP enzymes control reaction specificity by stereoelectronic control!
Pyridoxal phosphate (PLP)
example: alpha decarboxylase
substrate requires amino group to form Schiff-base with PLP
capable to cut behind alpha- and beta-C
Thiamine diphosphate
example: pyruvate decarboxylase
substrate does not require amino group
cuts exclusively behind alpha-C
Commonalities:
negative charge after cleavage is stabilized by positively charged N
double bond is formed and then protonated
Stereoelectronic control of PLP-dependent enzymes
Explain the catalytic strategy employed by mevalonate pyrophosphate decarboxylase!
Phosphate is a good leaving group and promotes elimination of CO2.
Name different suggested mechanisms for oritidine-5’-monophosphate decarboxylase! Which one(s) are supported or rules out by the experimental results?
ruled out by heavy atom kinetic isotope effects
no NMR proof found
not supported by active site structure
no heavy atom kinetic isotope effect
supported by active site structure
compatible with quantum chemical calculations
heavy atom kinetic isotope effect observable
* heavy atom kinetic isotope effect: in certain reactions, reaction with isotope is expected to be slower because it is heavier
Explain the mechanism of pyruvate formate lyase (PFL) and of the required PFL activase!
PFL mechanism
PFL activase mechanism
Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G734 of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes.
(https://doi.org/10.1073/pnas.0806640105)
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