Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase.

Evolution of dihydrofolate reductase (DHFR) has been studied using the enzyme from DHFR (ecDHFR) as a model, but less studies have used the enzyme from DHFR (hsDHFR). Each enzyme maintains a short and narrow distribution of hydride donor-acceptor distances (DAD) at the tunneling ready state (TRS). Evolution of the enzyme was previously studied in ecDHFR where three key sites were identified as important to the catalyzed reaction.

Caught in Action: X-ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog.

Methylation of 2-deoxyuridine-5'-monophosphate (dUMP) at the C5 position by the obligate dimeric thymidylate synthase (TSase) in the sole biosynthetic pathway to thymidine 5'-monophosphate (dTMP) proceeds by forming a covalent ternary complex with dUMP and cosubstrate 5,10-methylenetetrahydrofolate. The crystal structure of an analog of this intermediate gives important mechanistic insights but does not explain the half-of-the-sites activity of the enzyme. Recent experiments showed that the C5 proton and the catalytic Cys are eliminated in a concerted manner from the covalent ternary complex to produce a noncovalent bisubstrate intermediate.

Rigid Double-Stranded DNA Linkers for Single Molecule Enzyme-Drug Interaction Measurements Using Molecular Recognition Force Spectroscopy.

Single-molecule studies can reveal the distribution of states and interactions between ligand-enzyme complexes not accessible for most studies that measure a large ensemble average response of many molecules. Furthermore, in some biological applications, the information regarding the outliers, not the average of measured properties, can be more important. The high spatial and force resolution provided by atomic force microscopy (AFM) under physiological conditions has been utilized in this study to quantify the force-distance relations of enzyme-drug interactions.

Oscillatory Active-site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase.

Thermal motions of enzymes have been invoked to explain the temperature dependence of kinetic isotope effects (KIE) in enzyme-catalyzed hydride transfers. Formate dehydrogenase (FDH) from exhibits a temperature independent KIE that becomes temperature dependent upon mutation of hydrophobic residues in the active site. Ternary complexes of FDH that mimic the transition state structure allow investigation of how these mutations influence active-site dynamics.

Isotopic Labeling of Formate Dehydrogenase Perturbs the Protein Dynamics.

Isotope substitution of enzymes has become a means of addressing the participation of protein motions in enzyme-catalyzed reactions. The idea is that only the enzyme mass will be altered and not the electrostatics, so that the protein dynamics are essentially the same but at lower frequencies because of the mass change. In this study, we variably label all carbon atoms in formate dehydrogenase (FDH) with C, all nitrogen atoms with N, and all nonexchangeable hydrogen atoms with deuterium and investigate the impact that isotopic substitution has on the dynamics at the active site by two-dimensional infrared spectroscopy and compare with the measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs).