Direct evidence of catalytic heterogeneity in lactate dehydrogenase by temperature jump infrared spectroscopy

Michael J. Reddish, Huo Lei Peng, Hua Deng, Kunal S. Panwar, Robert Callender, R. Brian Dyer

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm-1, respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions.

Original languageEnglish (US)
Pages (from-to)10854-10862
Number of pages9
JournalJournal of Physical Chemistry B
Volume118
Issue number37
DOIs
StatePublished - Aug 22 2014

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lactates
dehydrogenases
L-Lactate Dehydrogenase
enzymes
Infrared spectroscopy
Spectrum Analysis
Enzymes
infrared spectroscopy
Temperature
pyruvates
Catalysis
Pyruvic Acid
Hydrides
Isotopes
hydrides
catalysis
isotopes
temperature
multiple access
Kinetics

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Materials Chemistry
  • Surfaces, Coatings and Films
  • Medicine(all)

Cite this

Direct evidence of catalytic heterogeneity in lactate dehydrogenase by temperature jump infrared spectroscopy. / Reddish, Michael J.; Peng, Huo Lei; Deng, Hua; Panwar, Kunal S.; Callender, Robert; Dyer, R. Brian.

In: Journal of Physical Chemistry B, Vol. 118, No. 37, 22.08.2014, p. 10854-10862.

Research output: Contribution to journalArticle

Reddish, Michael J. ; Peng, Huo Lei ; Deng, Hua ; Panwar, Kunal S. ; Callender, Robert ; Dyer, R. Brian. / Direct evidence of catalytic heterogeneity in lactate dehydrogenase by temperature jump infrared spectroscopy. In: Journal of Physical Chemistry B. 2014 ; Vol. 118, No. 37. pp. 10854-10862.
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