Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase

Abstract

Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis.T wo isotopic hybrids were prepared;o ne with the mobile N-terminal segment containing heavy isotopes ( 2 H, 13 C, 15 N) and the remainder of the protein with natural isotopic abundance,and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only aphysical step of catalysis,whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/ MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling,w hichi sd ifficult to obtain with other studies,a nd helps define the dynamic networks of intramolecular interactions central to enzyme catalysis.

Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies

“…3,4,37 Committor analysis also permits calculation of the barrier-crossing time for the hydride transfer reactions, which was the same for l- and h-DHFRs (Table 1). The computational data predict that ecDHFR is unlike other enzymes, 8-13 where altered enzyme mass has been demonstrated to influence the probability of barrier crossing. In ecDHFR, any vibrational motions altered by protein mass or temperature do not affect the TS or barrier-crossing parameters.…”

“…6,13 These investigations relied on steady-state k cat measurements at pH 9 and single-turnover kinetics at pH 7. It is unclear if the extracted kinetic constants reflect hydride transfer.…”

“…13 Those conclusions were drawn from differences in steady-state k cat and in the rate constants of fitting single-turnover kinetic data to a double-exponential equation. 6,13 However, the extracted rate constants might not report on hydride transfer chemistry at all temperatures since substrate KIEs were not reported. Transition state theory is often used to rationalize such results, but the application of traditional TS theory has been questioned in understanding protein dynamics in enzymatic reactions.…”

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis

“…A number of studies have illustrated the importance of enzyme motions and have suggested the presence of networks of vibrational modes that enable conformational sampling of productive substrate binding and/or reaction geometries (e.g. refs ). Modulating such a network would then have the potential to tune enzyme activity without altering the fine detail of the electrostatic environment at the active site, which is the key requirement for catalysis.…”

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

“…Protein-based approaches offer the ability to function efficiently under mild reaction conditions. 5,6 Transferase, [13][14][15] oxidoreductase, [16][17][18] ligase, 19,20 transpeptidase 2,4,[21][22][23][24][25][26][27] and (split-)intein [28][29][30] have been applied for protein bioconjugation. Nevertheless, adapting these enzymes or proteins for synthetic applications, which deviate from their natural function, oen results in technical issues.…”