| dc.contributor.advisor | Tittmann, Kai Prof. Dr. | |
| dc.contributor.author | Libuda, Fabienne | |
| dc.date.accessioned | 2018-08-22T09:17:57Z | |
| dc.date.available | 2018-08-22T09:17:57Z | |
| dc.date.issued | 2018-08-22 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-002E-E48C-9 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7024 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 572 | de |
| dc.title | Phosphoketolase - A mechanistic update | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Ficner, Ralf Prof. Dr. | |
| dc.date.examination | 2017-11-30 | |
| dc.description.abstracteng | The thiamine diphosphate (ThDP)-dependent enzyme phosphoketolase catalyzes the phosphorolytic
cleavage of fructose-6-phosphate and/or xylulose 5-phosphate under the generation of the
high-energy metabolite acetyl phosphate, as such playing a key role in the metabolism of lactic
acid bacteria and bifidobacteria. Phosphoketolase shows remarkable similarity to the prominent
ThDP-dependent enzyme transketolase and catalyzes as only known ThDP-dependent enzyme a
dehydration of its central alpha-carbanion/enamine intermediate.
Thanks to a small number of structural and functional studies, the phosphoketolase catalytic
mechanism is fairly well characterized. Dehydration of the alpha-carbanion/enamine cofactor adduct
(alpha,beta-dihydroxyethyl)-thiamine diphosphate (DHEThDP) produces an enol-acetyl-ThDP intermediate
that needs to undergo tautomerization to the corresponding keto-tautomer before a
nucleophilic attack from the second substrate phosphate leads to product formation.
In this thesis, a detailed analysis of the phosphoketolase reaction by kinetic and spectroscopic
techniques was carried out with special emphasis on the mechanism of acetyl-thiamine diphosphate
(AcThDP) tautomerization and the underlying chemical state of the post-dehydration intermediate
as well as potential carboligase side reactivity of the enzyme. The obtained results provide new
insights into the phosphoketolase reaction mechanism that suggest a mechanistic update for the
enol-keto-tautomerization of the post-dehydration intermediate AcThDP, give new information
on the similarities and differences of phosphoketolase and transketolase and led to the discovery
of a novel ThDP-catalyzed C-C bond forming side reaction.
Identification of a distinct UV-Vis absorbance signal associated to the phosphoketolase reaction
intermediate AcThDP provided the basis for a transient kinetic analysis of individual steps of the
phosphoketolase reaction. Additionally, a direct steady-state assay was established that allowed
determination of kinetic parameters for the phosphoketolase main reaction as well as for the
off-pathway hydrolysis of AcThDP to acetate that occurs in absence of the acyl-acceptor substrate
phosphate. The detailed kinetic examination revealed that the rate of enol-keto tautomerization
of AcThDP is increased by the presence of the acyl-acceptor substrate phosphate. Together
with results from spectroscopic and computational analysis of the phosphoketolase reaction, this
suggest a mechanism in which phosphate acts as substrate catalyst in the tautomerization from
enol- to keto-AcThDP. Direct catalytic involvement of phosphate provides coupling of the final acyl-transfer to the preceding tautomerization which avoids formation of the hydrolysis-susceptible
keto-AcThDP intermediate in the absence of an acceptor substrate.
The proposed mechanism for AcThDP tautomerization implies formation of an enolate-AcThDP
intermediate. Presence of this intermediate could be confirmed by trapping the enolate in a
carboligation reaction with formaldehyde. The side reaction between phosphoketolase bound
enolate-AcThDP and the aldehyde acceptor represents a novel type of ThDP-catalyzed C-C bond
forming reaction as it not originates from the central alpha-carbanion/enamine intermediate.
Generation of a "transketolase-like" phosphoketolase variant could not induce transketolase
activity, but showed the importance of three phosphoketolase-specific active side residues for the
phosphoketolase reaction mechanism. | de |
| dc.contributor.coReferee | Mata, Ricardo Prof. Dr. | |
| dc.subject.eng | Phosphoketolase | de |
| dc.subject.eng | Thiamin diphosphate | de |
| dc.subject.eng | Biocatalysis | de |
| dc.subject.eng | ThDP-dependent enzymes | de |
| dc.subject.eng | Transketolase | de |
| dc.subject.eng | Enzyme catalysis | de |
| dc.subject.eng | Carboligation | de |
| dc.subject.eng | Catalytic mechanism | de |
| dc.subject.eng | AcThDP | de |
| dc.subject.eng | Enolate-Intermediate | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-002E-E48C-9-0 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 1030406472 | |