Structural enzyme kinetics: studies on Chlorella DNA ligase

Samenvatting

Enzymology is a branch of biological chemistry aimed to study the molecular mechanisms of biocatalysis. Enzymatic reaction is a complex process that often involves homo- and heteromer enzyme cooperativity, inter- and intra-molecular electron transfer, multiple substrates, products, (in)organic cofactors, and proceeds via a large number of transient reaction intermediates. To understand in detail this rapid multi-component process, it is essential to resolve the catalytic reaction dynamics – the structural changes, which the enzyme/substrate undergo during the reaction turnover.
So far, to the applicant’s opinion, enzymology is lacking efficient experimental means to study the structural enzyme kinetics. The aim of this project is to lay down the foundation in this field, combining a rapid mixing-and-sampling technique (freeze-quench) with resonance spectroscopy (solid-state MAS NMR) for the analysis of the 3D-structures of transient reaction intermediates.
The experimental strategy will be used as follows.
- the enzymatic reaction will be initiated by a rapid mixing of the reaction components.
- the reaction intermediates will be isolated by a rapid freezing/hyperquenching.
- 3D structures of isolated intermediates will be analyzed using low-temperature solid-state MAS NMR spectroscopy.
The ultimate challenge of this project is to render a 3D-animation of the enzyme-catalyzed chemical reaction.
As a model system, binding of nicked dsDNA to DNA ligase from Chlorella virus PBCV-1 will be studied, selected for the following reasons.
- the known crystal structure of this enzyme will be used as a reference point in MAS NMR analysis.
- the ligase undergoes large conformational changes during catalysis: the intermediates are expected to have markedly different 3D structures.
- the molecular mechanism of DNA recognition by ligase is unknown. Structural studies of ligase-DNA interactions will bear scientific novelty in the field of DNA recognition and repair.

KEYWORDS: structural kinetics, transient state, structure-function correlation analysis

Kenmerken

Projectnummer

700.53.406

Hoofdaanvrager

Dr. A.V. Cherepanov

Verbonden aan

Goethe-Universität Frankfurt am Main

Uitvoerders

Dr. A.V. Cherepanov

Looptijd

01/04/2004 tot 12/11/2007