Enzymes are important macromolecules that speed up the rate of reactions such as in photosynthesis, respiration and protein synthesis and the metabolism process in the cells. Enzymes are known as biological catalysts because they lower down the activation energy while remained unchanged and can be reused after the reaction.

MoeZ is an enzyme that activates the sulfur carrier proteins by forming the C-S bond to deliver sulfur in the synthetic process of a 2-thiosugar in Amycolatopsis orientailis subsp. vinearia. This enzyme contains an E1-like adenylation domain and a rhodanese domain which are believed to catalyze a two-step reaction. However, the exact catalytic mechanism of how MoeZ activates the sulfur carrier proteins (MoaD₂, CysO, ThiS) is not clear. In this thesis, we prepared Mo...[ Read more ]

Enzymes are important macromolecules that speed up the rate of reactions such as in photosynthesis, respiration and protein synthesis and the metabolism process in the cells. Enzymes are known as biological catalysts because they lower down the activation energy while remained unchanged and can be reused after the reaction.

MoeZ is an enzyme that activates the sulfur carrier proteins by forming the C-S bond to deliver sulfur in the synthetic process of a 2-thiosugar in Amycolatopsis orientailis subsp. vinearia. This enzyme contains an E1-like adenylation domain and a rhodanese domain which are believed to catalyze a two-step reaction. However, the exact catalytic mechanism of how MoeZ activates the sulfur carrier proteins (MoaD₂, CysO, ThiS) is not clear. In this thesis, we prepared MoeZ and its sulfur carrier proteins and characterized their interaction by using various biological methods. MoeZ was found to interact with the sulfur carrier protein CysO and MoaD₂ in an ATP-dependent manner by native PAGE, the gel filtration profiling and MALDI-TOF analysis. On this basis, we attempted to co-crystallize the MoeZ-CysO and the MoeZ-MoaD₂ complex. Also, MoeZ was found to interact with ThiS to form a covalent adduct which was detected by SDS-PAGE but the MoeZ and ThiS were not co-eluted in the gel filtration process. We also intended to co-crystallize MoeZ with the sulfur carrier proteins in the presence of ATP to gain insights into its catalytic mechanism. Unfortunately, it was hard to obtain good crystals with satisfactory X-ray diffraction. The first part of the thesis presents the results of MoeZ crystallization and biochemical interaction between MoeZ and the sulfur carrier proteins.

In the second part, we expressed E. coli HPII and the activity, stability and heme concentration of purified HPII has been characterized. The total turn-over number of E. coli catalase HPII has been tested successfully which set a basis of future mutagenesis and structure studies. The total turn-over number has been tested which proved our hypothesis that the enzyme has a catalytic limit. Moreover, the total turn-over number showed no significant variation as temperature and pH changes, thus the total turn-over number should be a characteristic property of enzyme which is not be affected by environmental conditions. Secondly, by examining the UV spectrum, CD spectrum and desalting column elution profile of HPII before and after inactivation, we found that the inactivation of HPII was due to secondary structure collapse, heme group lost and denaturation. In this part of the thesis, we concluded that although the enzyme is a high-efficiency molecule, it also has a catalytic limit.