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 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.

Selective Aptamer Modification of Au Surfaces in a Microelectrode Sensor Array for Simultaneous Detection of Multiple Analytes

“…This result is in reasonable agreement with previously reported literature for this ATP E-AB sensor on a 2 mm Au disk electrode . It was also observed that the peak shifted to a more positive potential with increasing ATP concentration (Figure a), which was attributed to a decrease in the solution pH. , …”

A Review of Methods to Determine Viability, Vitality, and Metabolic Rates in Microbiology

“…The exact process leading to ATP hydrolysis remains unclear; still, much care has to be taken when analyzing environmental samples and drawing conclusions about activity (or biomass). Also, for all types of samples, the pH must be controlled with care, as it has been shown to affect the results ( Posimo et al, 2014 ; Šimèíková and Heneberg, 2019 ).…”

Exploring monocarboxylate transporter inhibition for cancer treatment

“…This last experiment shows that the NBC increases intracellular buffering capacity by importing bicarbonate and this permits an increased lactate influx without decreasing intracellular pH. This evidence shows that the pHtome works in a coordinated manner [223].…”

“…The optimum pH for glucokinase and hexokinase II was originally thought to be in the low alkaline level. However, it is now known to be more alkaline, in the range of 8.5–8.7 [ 224 ] for glucokinase. For other enzymes, their pH optimums are also alkaline, 7.8 for hexokinase I and 8.1 for hexokinase III [ 225 ].…”