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Effects of Isotopic Substitution in Enzyme and Co‐factor on Enzyme Catalyzed Hydride Transfer
Author(s) -
Ranasinghe Chethya,
Pagano Philip,
Guo Qi,
Cheatum Christopher,
Kohen Am
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.764.1
Subject(s) - chemistry , kinetic isotope effect , cofactor , formate , nad+ kinase , hydride , formate dehydrogenase , nicotinamide adenine dinucleotide , nicotinamide , enzyme , stereochemistry , photochemistry , catalysis , deuterium , metal , atomic physics , biochemistry , organic chemistry , physics
Enzyme dynamics range from seconds to femtoseconds. Previous computational, theoretical, and experimental studies have shown an imperative role of slow enzyme dynamics in its function. It is equally important to unravel the origin of fast enzyme dynamics, which are more likely to play a role in catalyzing chemical bond cleavage. Isotopically labeled (heavy) enzymes and subsequently heavy cofactors were employed to experimentally study the role of fast dynamics. Here, we used nicotinamide adenine dinucleotide (NAD + ) dependent formate dehydrogenase (FDH, EC 1.2.1.2) as the model system. Heavy enzyme studies were carried out with 13 C; 15 N and 13 C; 15 N; 2 H labeled FDHs. Temperature dependence of kinetic isotope effects (KIE) were compared between light and heavy FDHs. The results showed that perturbation of fast enzyme dynamics by mass modulation, alters the chemical step at the tunneling ready state (TRS, a diffused transition state) of the catalyzed hydride transfer. Studies are underway to examine similar trend using heavy cofactor (NAD + ). Temperature dependence of KIE would give insight to the extent of contribution of the ring motions in sampling the donor‐acceptor distances at the TRS. In addition to the kinetic measurements, vibrational spectroscopy will be used to test the effect of heavy protein and cofactor on dynamics at the enzyme's transition state. Azide ion, which is a transition state analog of the FDH catalyzed reaction, is a good IR chromophore. This enables the use of vibrational spectroscopic methods such as two dimensional IR (2D‐IR), to directly probe the femtosecond‐picosecond timescale motions in the enzyme active site. Together, these studies should shed light on the molecular origin of fast motions that enhance catalytic efficiency of enzymes. Support or Funding Information NIH GM065368 and GM79368, NSF CHE‐1149023, BSF 2012340