Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase

Miriam Gulotta, Hua Deng, Hong Deng, R. Brian Dyer, Robert Callender

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The motions of key residues at the substrate binding site of lactate dehydrogenase (LDH) were probed on the 10 ns to 10 ms time scale using laser-induced temperature-jump relaxation spectroscopy employing both UV fluorescence and isotope-edited IR absorption spectroscopy as structural probes. The dynamics of the mobile loop, which closes over the active site and is important for catalysis and binding, were characterized by studies of the inhibitor oxamate binding to the LDH/NADH binary complex monitoring the changes in emission of bound NADH. The bound NAD-pyruvate adduct, whose pyruvate moiety likely interacts with the same residues that interact with pyruvate in its ternary complex with LDH, served as a probe for any relative motions of active site residues against the substrate. The frequencies of its C=O stretch and -COO- antisymmetric stretch shift substantially should any relative motion of the polar moieties at the active site (His-195, Asp-168, Arg-109, and Arg-171) occur. The dynamics associated with loop closure are observed to involve several steps with motions from 1 to 300 μs. Apart from the "melting" of a few residues on the protein's surface, no kinetics were observed on any time scale in experiments of the bound NAD-pyr adduct although the measurements were made with a high degree of accuracy, even for final temperatures close to the unfolding transition of the protein. This is contrary to simple physical considerations and models. These results show that, once a productive protein/ substrate complex is formed, the binding pocket is very rigid with very little, if any, motion apart from the mobile loop. The results also show that loop opening involves concomitant movement of the substrate out of the binding pocket.

Original languageEnglish (US)
Pages (from-to)3353-3363
Number of pages11
JournalBiochemistry
Volume41
Issue number10
DOIs
StatePublished - Mar 12 2002

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Catalysis
L-Lactate Dehydrogenase
NAD
Substrates
Catalytic Domain
Pyruvic Acid
Spectrum Analysis
Protein Unfolding
Absorption spectroscopy
Temperature
Isotopes
Infrared spectroscopy
Membrane Proteins
Proteins
Melting
Fluorescence
Binding Sites
Freezing
Spectroscopy
Lasers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase. / Gulotta, Miriam; Deng, Hua; Deng, Hong; Dyer, R. Brian; Callender, Robert.

In: Biochemistry, Vol. 41, No. 10, 12.03.2002, p. 3353-3363.

Research output: Contribution to journalArticle

Gulotta, Miriam ; Deng, Hua ; Deng, Hong ; Dyer, R. Brian ; Callender, Robert. / Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase. In: Biochemistry. 2002 ; Vol. 41, No. 10. pp. 3353-3363.
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