Hydrogen Tunneling in Enzyme Reactions

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

There are now close to 20 documented examples of a role for quantum effects in enzyme catalyzed C-H activation processes. Insights from the ongoing work on H-tunneling in enzymes include the recognition that enzymes modify the entire reaction barrier shape (i.e., reaction barrier width as well as height) and that protein dynamics may play a role in the bond cleavage events catalyzed by enzymes. Further investigations are to be focused on establishing a firmer and more detailed relationship between protein dynamics and H-tunneling. Several hydride transfer reactions will be studied, catalyzed by a high temperature alcohol dehydrogenase (ht-ADH) with homology to the mesophilic horse liver alcohol dehydrogenase and a thermophilic dihydrofolate reductase (ht-DHFR) with significant homology to mesophilic DHFR. In the case of ht-ADH, studies are in progress to solve the three dimensional structure for this enzyme. This structural information will guide site specific mutagenesis experiments to analyze the effect of protein side chains on catalysis. In particular, the effects at elevated temperatures where tunneling is dominant and at reduced temperatures where H-transfer becomes more classical will be analyzed. Since the ht-ADH is too large for a study of dynamics by NMR, H/D exchange, followed by limited proteolysis and mass spectrometric analysis will be pursued to map out protein flexibility. This laboratory has recently succeeded in cloning and over-expressing a ht-DHFR from B. stearothermophilis. This protein will be analyzed for the contribution of tunneling to H-transfer as a function of temperature. In parallel studies, this small protein (19 KDa) will be studied by NMR (as a probe of local dynamics) and by FT-IR H/D exchange (as a probe of global flexibility).