A sensitive coupled enzyme assay for measuring kinase and ATPase kinetics using ADP-specific hexokinase
File(s)
Published version
OA Location
Author(s)
McFarlane, Ciaran
Murray, James
Type
Journal Article
Abstract
Kinases and ATPases perform essential biological functions in metabolism and regulation.
Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a
detectable product. For most assay systems the ATP concentration during the reaction is unknown,
compromising the precision of the assay.
Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon Thermococcus litoralis
the protocol outlined here allows real time coupling of ATP consumption to downstream signal change
enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose6-phosphate dehydrogenase to reduce NAD+ to NADH which can be measured at 340 nm. We have
shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable
background from ATP.
Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a
detectable product. For most assay systems the ATP concentration during the reaction is unknown,
compromising the precision of the assay.
Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon Thermococcus litoralis
the protocol outlined here allows real time coupling of ATP consumption to downstream signal change
enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose6-phosphate dehydrogenase to reduce NAD+ to NADH which can be measured at 340 nm. We have
shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable
background from ATP.
Date Issued
2020-05-05
Date Acceptance
2020-05-01
Citation
Bio-protocol, 2020, 10 (9), pp.1-10
ISSN
2331-8325
Publisher
Bio-Protocol
Start Page
1
End Page
10
Journal / Book Title
Bio-protocol
Volume
10
Issue
9
Copyright Statement
© 2020 The Authors; exclusive licensee Bio-protocol LLC.
Identifier
https://bio-protocol.org/e3599
Publication Status
Published
Date Publish Online
2020-05-05