Energy landscape of the michaelis complex of lactate dehydrogenase: Relationship to catalytic mechanism

Huo Lei Peng, Hua Deng, R. Brian Dyer, Robert Callender

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the "live" reaction mixture (LDH·NADH·pyruvate LDH·NAD+·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate's C 2=O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2î - O group and active site residues, as shown by the observation of at least four C2=O stretch bands indicating varying degrees of C2=O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution.

Original languageEnglish (US)
Pages (from-to)1849-1857
Number of pages9
JournalBiochemistry
Volume53
Issue number11
DOIs
StatePublished - Mar 25 2014

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Pyruvic Acid
L-Lactate Dehydrogenase
NAD
Lactic Acid
Observation
Niacinamide
Fourier Transform Infrared Spectroscopy
Mutant Proteins
Enzymes
Structure-Activity Relationship
Catalysis
Isotopes
Hydrogen
Catalytic Domain
Hydrogen bonds
Polarization
Population
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Energy landscape of the michaelis complex of lactate dehydrogenase : Relationship to catalytic mechanism. / Peng, Huo Lei; Deng, Hua; Dyer, R. Brian; Callender, Robert.

In: Biochemistry, Vol. 53, No. 11, 25.03.2014, p. 1849-1857.

Research output: Contribution to journalArticle

Peng, Huo Lei ; Deng, Hua ; Dyer, R. Brian ; Callender, Robert. / Energy landscape of the michaelis complex of lactate dehydrogenase : Relationship to catalytic mechanism. In: Biochemistry. 2014 ; Vol. 53, No. 11. pp. 1849-1857.
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