The approach to the Michaelis complex in lactate dehydrogenase: The substrate binding pathway

Sebastian McClendon, Nick Zhadin, Robert Callender

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His195 and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme Kd values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, ∼10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.

Original languageEnglish (US)
Pages (from-to)2024-2032
Number of pages9
JournalBiophysical Journal
Volume89
Issue number3
DOIs
StatePublished - Sep 2005

Fingerprint

L-Lactate Dehydrogenase
Hydrogen Bonding
NAD
Niacinamide
Enzymes
Thermodynamics
Protein Binding
Mental Competency
Freezing
Catalytic Domain
Spectrum Analysis
Membrane Proteins
Lasers
Binding Sites
Temperature
Water
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

The approach to the Michaelis complex in lactate dehydrogenase : The substrate binding pathway. / McClendon, Sebastian; Zhadin, Nick; Callender, Robert.

In: Biophysical Journal, Vol. 89, No. 3, 09.2005, p. 2024-2032.

Research output: Contribution to journalArticle

@article{97a9d566a839469c8dabedc7bf451978,
title = "The approach to the Michaelis complex in lactate dehydrogenase: The substrate binding pathway",
abstract = "We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His195 and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme Kd values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, ∼10-15{\%}, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.",
author = "Sebastian McClendon and Nick Zhadin and Robert Callender",
year = "2005",
month = "9",
doi = "10.1529/biophysj.105.062604",
language = "English (US)",
volume = "89",
pages = "2024--2032",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "3",

}

TY - JOUR

T1 - The approach to the Michaelis complex in lactate dehydrogenase

T2 - The substrate binding pathway

AU - McClendon, Sebastian

AU - Zhadin, Nick

AU - Callender, Robert

PY - 2005/9

Y1 - 2005/9

N2 - We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His195 and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme Kd values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, ∼10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.

AB - We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His195 and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme Kd values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, ∼10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.

UR - http://www.scopus.com/inward/record.url?scp=23244457509&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=23244457509&partnerID=8YFLogxK

U2 - 10.1529/biophysj.105.062604

DO - 10.1529/biophysj.105.062604

M3 - Article

C2 - 15980172

AN - SCOPUS:23244457509

VL - 89

SP - 2024

EP - 2032

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 3

ER -