The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

Abstract

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) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) 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 5–45 °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 yield 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.

Protein Mass Effects on Formate Dehydrogenase

“…The observed change in the (intrinsic) KIEs can be achieved in a number of ways besides isotopic labeling of protein and/or cofactor; 33 eg: hydrostatic pressure, 62 site-directed mutagenesis 63,64 and alterations in protein solvation, 65 use of un-natural (alternate) substrates, 66,67 or even species variation across the evolutionary timeline (eg: variation in the intrinsic KIE values of ecDHFR and hsDHFR although both show temperature independence) 28 etc. The interpretation of the temperature dependence of KIEs, however, is similar in all of these instances, i.e.…”

“…21,23 It was suggested that these studies reflect a decrease in the frequency of fs motions that are linked to barrier crossing and that the outcomes support the concept of “Born Oppenheimer” enzymes. Studies on bacterial and human dihydrofolate reductase (DHFR) 14,28 revealed no effect of the heavy DHFR on intrinsic KIEs (at physiological temperatures); however, the mass alteration affected protein-ligand interactions, which are expected to take place at a slower timescale, and may indicate alteration of the protein potential surface.…”

Examinations of the Chemical Step in Enzyme Catalysis

“…It seems that the experimental studies using KIE int agree well with the first but did not test the second theoretical approach. Additionally, TPS calculatins of the human DHFR (hsDHFR) predicted significant PPVs, but KIE int for both light and heavy hsDHFR where identical throughout the temperature range (Francis, Sapienza, Lee, & Kohen, 2016), falling to support the theoretical prediction. Similarly, in a different TPS study of liver alcohol dehydrogenase, it was predicted that the valine residues at position 203 and 207 facilitate enzyme catalysis as part of the PPV (Caratzoulas, Mincer, & Schwartz, 2002).…”

Trendbericht Physikalische Chemie 2017: Atomare und molekulare Tunnelprozesse

“…Programmsysteme der klassischen MD, damit sie auf biomolekulare Tunnelprozesse anwendbar sind. 34,[41][42][43][44][45][46][47][48][49][50][51][52] Ein weiteres Verfahren, die Quantendynamik vielatomiger Moleküle zu beschreiben, beruht auf der MCTDH-Methode (Multi Configuration Time Dependent Hartee). [53][54][55][56] Das Multimode-Package wurde ebenfalls auf Tunnelprobleme angewandt.…”