Open AccessEffect of Protein Isotope Labeling on the Catalytic Mechanism of Lactate Dehydrogenase
Author(s) -
Tsuyoshi Egawa,
Hua Deng,
Eric P. Chang,
Robert Callender
Publication year - 2019
Publication title -
the journal of physical chemistry. b
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 392
eISSN - 1520-6106
pISSN - 1520-5207
DOI - 10.1021/acs.jpcb.9b08656
Subject(s) - chemistry , kinetic isotope effect , microsecond , enzyme kinetics , lactate dehydrogenase , spectroscopy , isotopic labeling , catalysis , reaction coordinate , protein dynamics , biophysics , enzyme , protein structure , active site , deuterium , computational chemistry , biochemistry , biology , atomic physics , physics , organic chemistry , astronomy , quantum mechanics
We investigate how isotopic labeling of the enzyme lactate dehydrogenase (LDH) affects its function. LDH is of special interest because there exists a line of residues spanning the protein that are involved in the transition state (TS) of the chemical reaction coordinate (so-called promoting vibration). Hence, studies have been carried out on this protein (as well as others) using labeled protein (so-called heavy protein) along with measurements of single turnover k cat yielding a KIE (= k cat light / k cat heavy ) aimed at understanding the effect of labeling generally and more specifically this line of residues. Here, it is shown that 13 C, 15 N, and 2 H atom labeling of hhLDH (human heart) affects its internal structure which in turn affects its dynamics and catalytic mechanism. Spectral studies employing advanced FTIR difference spectroscopy show that the height of the electronic potential surface of the TS is lowered (probably by ground state destabilization) by labeling. Moreover, laser-induced T-jump relaxation kinetic spectroscopy shows that the microsecond to millisecond nuclear motions internal to the protein are affected by labeling. While the effects are small, they are sufficient to contribute to the observed KIE values as well or even more than promoting vibration effects.