Reaction Mechanism and Allosteric Regulation of Class Ia Ribonucleotide Reductase from Escherichia coli
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Ribonculeotide reductase (RNR) plays a key role in catalysing a reaction that provides all living organisms with building blocks for DNA. The RNR enzyme catalyses the conversion of ribonucleotides to their corresponding deoxyribonucleotides, by using free radical chemistry. The class Ia enzyme from Escherichia coli is composed of two components, R1 and R2 proteins. Both components are required for the catalytic reaction. The R2 protein contains a stable tyrosyl free radical, which is directly involved in the catalysis. The R1 protein, contains the active site and two different allosteric sites, which are responsible for regulating the enzyme activity and its substrate specificity.
An evolutionary conserved active site residue, Asn437, was studied. Employing site directed mutagensis it was shown that the Asn437 is essential for the catalytic reaction. Also using biophysical methods, it was shown that the Asn437 residue participates in the later part of the catalytic reaction.
By using protein engineering, two conserved overall activity residues His59 and His88 were shown to be involved in the allosteric regulation of enzymatic activity. Furthermore, it was shown by biosensor technique (BIAcore) that the His59 and His88 participate in a communication network that regulates the binding affinity between the R1 and R2 components in response to the presence of certain allosteric effectors.
The influence of the allosteric effectors and substrate nucleotides on the wild type R1/R2 interaction was investigated using biosensor technique. The presence of allosteric effectors strengthened the affinity between the components R1 and R2 as compared to the affinity in the absence of effectors. Interestingly, when both allosteric effectors and substrate nucleotides were present, the affinity between the R1 and R2 components was noticeably stronger as compared to the situations where only the allosteric effector was present.
Finally, the residues located in the interaction area of the dimeric R1 protein from E. coli were studied. The aim was to understand their importance for the formation of the active dimeric form of R1. Interestingly, slight alterations of the chemical properties of single side chains resulted in drastic changes in dimer stability, indicating the importance of their interaction ability for dimer formation.
Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik , 2004. , 69 p.
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:su:diva-34ISBN: 91-7265-810-XOAI: oai:DiVA.org:su-34DiVA: diva2:192679
2004-02-27, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00
Jonsson, Bengt-Harald, Professor
Sjöberg, Britt-Marie, Professor