Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays

Abstract Adenosine triphosphate (ATP) is a crucial substrate and energy source commonly used in enzyme reactions. However, we demonstrated that the addition of this acidic compound to enzyme assay buffers can serve as a source of unnoticed pH changes. Even relatively low concentrations of ATP (up to...

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Autores principales:	Daniela Šimčíková, Petr Heneberg
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2019
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3d1ae72d90784320879ed8194e44556c
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spelling	oai:doaj.org-article:3d1ae72d90784320879ed8194e44556c2021-12-02T15:07:53ZIdentification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays10.1038/s41598-019-47883-12045-2322https://doaj.org/article/3d1ae72d90784320879ed8194e44556c2019-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-019-47883-1https://doaj.org/toc/2045-2322Abstract Adenosine triphosphate (ATP) is a crucial substrate and energy source commonly used in enzyme reactions. However, we demonstrated that the addition of this acidic compound to enzyme assay buffers can serve as a source of unnoticed pH changes. Even relatively low concentrations of ATP (up to 5 mM) shifted pH of reaction mixtures to acidic values. For example, Tris buffer lost buffering capacity at pH 7.46 by adding ATP at a concentration higher than 2 mM. In addition to the buffering capacity, the pH shifts differed with respect to the buffer concentration. High ATP concentrations are commonly used in hexokinase assays. We demonstrated how the presence of ATP affects pH of widely used enzyme assay buffers and inversely affected K M of human hexokinase 2 and S 0.5 of human glucokinase. The pH optimum of human glucokinase was never reported before. We found that previously reported optimum of mammalian glucokinase was incorrect, affected by the ATP-induced pH shifts. The pH optimum of human glucokinase is at pH 8.5–8.7. Suggested is the full disclosure of reaction conditions, including the measurement of pH of the whole reaction mixtures instead of measuring pH prior to the addition of all the components.Daniela ŠimčíkováPetr HenebergNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 9, Iss 1, Pp 1-6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daniela Šimčíková Petr Heneberg Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays
description	Abstract Adenosine triphosphate (ATP) is a crucial substrate and energy source commonly used in enzyme reactions. However, we demonstrated that the addition of this acidic compound to enzyme assay buffers can serve as a source of unnoticed pH changes. Even relatively low concentrations of ATP (up to 5 mM) shifted pH of reaction mixtures to acidic values. For example, Tris buffer lost buffering capacity at pH 7.46 by adding ATP at a concentration higher than 2 mM. In addition to the buffering capacity, the pH shifts differed with respect to the buffer concentration. High ATP concentrations are commonly used in hexokinase assays. We demonstrated how the presence of ATP affects pH of widely used enzyme assay buffers and inversely affected K M of human hexokinase 2 and S 0.5 of human glucokinase. The pH optimum of human glucokinase was never reported before. We found that previously reported optimum of mammalian glucokinase was incorrect, affected by the ATP-induced pH shifts. The pH optimum of human glucokinase is at pH 8.5–8.7. Suggested is the full disclosure of reaction conditions, including the measurement of pH of the whole reaction mixtures instead of measuring pH prior to the addition of all the components.
format	article
author	Daniela Šimčíková Petr Heneberg
author_facet	Daniela Šimčíková Petr Heneberg
author_sort	Daniela Šimčíková
title	Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays
title_short	Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays
title_full	Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays
title_fullStr	Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays
title_full_unstemmed	Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays
title_sort	identification of alkaline ph optimum of human glucokinase because of atp-mediated bias correction in outcomes of enzyme assays
publisher	Nature Portfolio
publishDate	2019
url	https://doaj.org/article/3d1ae72d90784320879ed8194e44556c
work_keys_str_mv	AT danielasimcikova identificationofalkalinephoptimumofhumanglucokinasebecauseofatpmediatedbiascorrectioninoutcomesofenzymeassays AT petrheneberg identificationofalkalinephoptimumofhumanglucokinasebecauseofatpmediatedbiascorrectioninoutcomesofenzymeassays
_version_	1718388309116321792

Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays

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