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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11120%2F19%3A43918501" target="_blank" >RIV/00216208:11120/19:43918501 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1038/s41598-019-47883-1" target="_blank" >https://doi.org/10.1038/s41598-019-47883-1</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1038/s41598-019-47883-1" target="_blank" >10.1038/s41598-019-47883-1</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

30202 - Endocrinology and metabolism (including diabetes, hormones)

Projekt

<a href="/cs/project/GJ15-03834Y" target="_blank" >GJ15-03834Y: Mechanismy vzniku, progrese a terapie monogenních typů diabetu</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Scientific Reports

ISSN

2045-2322

e-ISSN

—

Svazek periodika

9

Číslo periodika v rámci svazku

August

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

6

Strana od-do

"Article 11422"

Kód UT WoS článku

000478864200013

EID výsledku v databázi Scopus

2-s2.0-85070315860