Effect of Protein Isotope Labeling on the Catalytic Mechanism of Lactate Dehydrogenase

Tsuyoshi Egawa, Hua Deng, Eric Chang, Robert Callender

Research output: Contribution to journalArticle

Abstract

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 kcat yielding a KIE (=kcat light/kcat heavy) aimed at understanding the effect of labeling generally and more specifically this line of residues. Here, it is shown that 13C, 15N, and 2H 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.

Original languageEnglish (US)
JournalJournal of Physical Chemistry B
DOIs
StateAccepted/In press - Jan 1 2019

Fingerprint

lactates
dehydrogenases
L-Lactate Dehydrogenase
Isotopes
Labeling
marking
isotopes
proteins
Proteins
vibration effects
Spectroscopy
isotopic labeling
destabilization
Surface potential
Ground state
spectroscopy
enzymes
Chemical reactions
chemical reactions
Enzymes

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

Cite this

Effect of Protein Isotope Labeling on the Catalytic Mechanism of Lactate Dehydrogenase. / Egawa, Tsuyoshi; Deng, Hua; Chang, Eric; Callender, Robert.

In: Journal of Physical Chemistry B, 01.01.2019.

Research output: Contribution to journalArticle

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