Precise, facile initial rate measurements

Qingxiu Tang, Thomas S. Leyh

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Progress curve analysis has been used sparingly in studies of enzyme-catalyzed reactions due largely to the complexity of the integrated rate expressions used in data analysis. Using an experimental design that simplifies the analysis, the advantages and limitations of progress curve experiments are explored in a study of four different enzyme-catalyzed reactions. The approach involves relatively simple protocols, requires 20-25% of the materials, and provides 10- to 20-fold signal enhancements compared to analogous initial rate studies. Product inhibition, which complicates integrated rate analysis, was circumvented using cloned, purified enzymes that remove the products and draw the reaction forward. The resulting progress curves can be transformed into the equivalent of thousands of initial rate and [S] measurements and, due to the absence of product inhibition, are plotted in the familiar, linear double-reciprocal format. Allowing product to accumulate during a reaction produces a continuously changig substrate/product ratio that can be used as the basis for obtaining product inhibition constants and to distinguish among the three classical inhibition mechanisms. Algebraic models describing the double-reciprocal patterns obtained from such inhibition studies are presented. The virtual continuum of substrate concentrations that occurs during a progress curve experiment provides a nearly errorless set of relative concentrations that results in remarkably precise data; kinetic constant standard deviations are on the order of 0.5%.

Original languageEnglish (US)
Pages (from-to)16131-16136
Number of pages6
JournalJournal of Physical Chemistry B
Volume114
Issue number49
DOIs
StatePublished - Dec 16 2010

Fingerprint

Enzymes
products
enzymes
Substrates
curves
Design of experiments
Experiments
Kinetics
format
standard deviation
continuums
augmentation
kinetics

ASJC Scopus subject areas

  • Surfaces, Coatings and Films
  • Physical and Theoretical Chemistry
  • Materials Chemistry

Cite this

Precise, facile initial rate measurements. / Tang, Qingxiu; Leyh, Thomas S.

In: Journal of Physical Chemistry B, Vol. 114, No. 49, 16.12.2010, p. 16131-16136.

Research output: Contribution to journalArticle

Tang, Qingxiu ; Leyh, Thomas S. / Precise, facile initial rate measurements. In: Journal of Physical Chemistry B. 2010 ; Vol. 114, No. 49. pp. 16131-16136.
@article{87635d6a5d30483ca7361777e32a8c23,
title = "Precise, facile initial rate measurements",
abstract = "Progress curve analysis has been used sparingly in studies of enzyme-catalyzed reactions due largely to the complexity of the integrated rate expressions used in data analysis. Using an experimental design that simplifies the analysis, the advantages and limitations of progress curve experiments are explored in a study of four different enzyme-catalyzed reactions. The approach involves relatively simple protocols, requires 20-25{\%} of the materials, and provides 10- to 20-fold signal enhancements compared to analogous initial rate studies. Product inhibition, which complicates integrated rate analysis, was circumvented using cloned, purified enzymes that remove the products and draw the reaction forward. The resulting progress curves can be transformed into the equivalent of thousands of initial rate and [S] measurements and, due to the absence of product inhibition, are plotted in the familiar, linear double-reciprocal format. Allowing product to accumulate during a reaction produces a continuously changig substrate/product ratio that can be used as the basis for obtaining product inhibition constants and to distinguish among the three classical inhibition mechanisms. Algebraic models describing the double-reciprocal patterns obtained from such inhibition studies are presented. The virtual continuum of substrate concentrations that occurs during a progress curve experiment provides a nearly errorless set of relative concentrations that results in remarkably precise data; kinetic constant standard deviations are on the order of 0.5{\%}.",
author = "Qingxiu Tang and Leyh, {Thomas S.}",
year = "2010",
month = "12",
day = "16",
doi = "10.1021/jp1055528",
language = "English (US)",
volume = "114",
pages = "16131--16136",
journal = "Journal of Physical Chemistry B Materials",
issn = "1520-6106",
publisher = "American Chemical Society",
number = "49",

}

TY - JOUR

T1 - Precise, facile initial rate measurements

AU - Tang, Qingxiu

AU - Leyh, Thomas S.

PY - 2010/12/16

Y1 - 2010/12/16

N2 - Progress curve analysis has been used sparingly in studies of enzyme-catalyzed reactions due largely to the complexity of the integrated rate expressions used in data analysis. Using an experimental design that simplifies the analysis, the advantages and limitations of progress curve experiments are explored in a study of four different enzyme-catalyzed reactions. The approach involves relatively simple protocols, requires 20-25% of the materials, and provides 10- to 20-fold signal enhancements compared to analogous initial rate studies. Product inhibition, which complicates integrated rate analysis, was circumvented using cloned, purified enzymes that remove the products and draw the reaction forward. The resulting progress curves can be transformed into the equivalent of thousands of initial rate and [S] measurements and, due to the absence of product inhibition, are plotted in the familiar, linear double-reciprocal format. Allowing product to accumulate during a reaction produces a continuously changig substrate/product ratio that can be used as the basis for obtaining product inhibition constants and to distinguish among the three classical inhibition mechanisms. Algebraic models describing the double-reciprocal patterns obtained from such inhibition studies are presented. The virtual continuum of substrate concentrations that occurs during a progress curve experiment provides a nearly errorless set of relative concentrations that results in remarkably precise data; kinetic constant standard deviations are on the order of 0.5%.

AB - Progress curve analysis has been used sparingly in studies of enzyme-catalyzed reactions due largely to the complexity of the integrated rate expressions used in data analysis. Using an experimental design that simplifies the analysis, the advantages and limitations of progress curve experiments are explored in a study of four different enzyme-catalyzed reactions. The approach involves relatively simple protocols, requires 20-25% of the materials, and provides 10- to 20-fold signal enhancements compared to analogous initial rate studies. Product inhibition, which complicates integrated rate analysis, was circumvented using cloned, purified enzymes that remove the products and draw the reaction forward. The resulting progress curves can be transformed into the equivalent of thousands of initial rate and [S] measurements and, due to the absence of product inhibition, are plotted in the familiar, linear double-reciprocal format. Allowing product to accumulate during a reaction produces a continuously changig substrate/product ratio that can be used as the basis for obtaining product inhibition constants and to distinguish among the three classical inhibition mechanisms. Algebraic models describing the double-reciprocal patterns obtained from such inhibition studies are presented. The virtual continuum of substrate concentrations that occurs during a progress curve experiment provides a nearly errorless set of relative concentrations that results in remarkably precise data; kinetic constant standard deviations are on the order of 0.5%.

UR - http://www.scopus.com/inward/record.url?scp=78650165000&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78650165000&partnerID=8YFLogxK

U2 - 10.1021/jp1055528

DO - 10.1021/jp1055528

M3 - Article

VL - 114

SP - 16131

EP - 16136

JO - Journal of Physical Chemistry B Materials

JF - Journal of Physical Chemistry B Materials

SN - 1520-6106

IS - 49

ER -