Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

Abstract

Much effort has been directed towards understanding the contributions of electrostatics and dynamics to protein function and especially to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct experimental probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biological catalysis. We observe that the stretching absorption frequency of (methyl-d 3 ) methionine carbon-deuterium bonds show an approximately linear dependence on solvent dielectric. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary analysis of (methyl-d 3 ) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics, and potentially, their contribution to catalysis.

Influence of Equatorial CH⋅⋅⋅O Interactions on Secondary Kinetic Isotope Effects for Methyl Transfer

“…The functional importance of CH⋅⋅⋅O hydrogen bonding in AdoMet‐dependent methyltransferases has been argued,7 but a link to KIEs has not been previously proposed. It is known, however, that CD bond stretching frequencies are sensitive to the local electric field within a protein environment 16. Klinman and co‐workers have reported T 3 KIEs on k cat / K m in human COMT: 0.791±0.012 for the wild‐type enzyme and 0.822±0.021 and 0.850±0.012 for its Y68F and Y68A mutants, respectively 2, 3.…”

Decomposition of Vibrational Shifts of Nitriles into Electrostatic and Hydrogen-Bonding Effects

“…While IR frequency shifts associated with various environments can be considered at a qualitative level (e.g., is the probe buried or on a solvent exposed surface?7,8), our goal has been to extract quantitative information on electric fields in proteins1,2. Although several studies have suggested a connection between observed vibrational band shifts and local electrostatic fields due to the organized environment around the probe3,6, in the absence of an independent experimental test it remains uncertain whether these shifts are due principally to electrostatic effects or are dominated by contributions from specific chemical interactions9, such as hydrogen bonds. We report herein a method for identifying and quantifying departures from an electrostatic mechanism for nitrile vibrational shifts that utilizes the relationship between IR frequency shifts and 13 C NMR chemical shifts and demonstrate its utility in a protein.…”

Efforts Toward the Direct Experimental Characterization of Enzyme Microenvironments: Tyrosine100 in Dihydrofolate Reductase

“…This latter approach has been applied to DHFR in a previous study wherein all but one methionine was mutagenized to leucine to allow for site-specific labeling. [23] In order to examine a residue such as Tyr100 in DHFR without the introduction of potentially perturbative mutations, we now report the use of a biosynthetic method to site-selectively incorporate a photocaged, deuterated amino acid, which after photolysis yields the site-selectively deuterated, but otherwise natural protein.…”