The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase
详细信息 查看全文
- 作者：Kevin Francis ; Paul J. Sapienza ; Andrew L. Lee ; Amnon Kohen
- 刊名：Biochemistry
- 出版年：2016
- 出版时间：February 23, 2016
- 年：2016
- 卷：55
- 期：7
- 页码：1100-1106
- 全文大小：341K
- ISSN：1520-4995
文摘
Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C–H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C–H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general.