Large scale dynamics of the michaelis complex in Bacillus stearothermophilus lactate dehydrogenase revealed by a single-tryptophan mutant study

Beining Nie, Hua Deng, Ruel Desamero, Robert Callender

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophilic lactate dehydrogenase, bsLDH·NADH· oxamate, were studied with site specific resolution by laser-induced temperature jump relaxation spectroscopy with a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the millisecond to microsecond time scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called "loop motion" is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on the catalytic mechanism of bsLDH is discussed.

Original languageEnglish (US)
Pages (from-to)1886-1892
Number of pages7
JournalBiochemistry
Volume52
Issue number11
DOIs
StatePublished - Mar 19 2013

ASJC Scopus subject areas

  • Biochemistry

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