Protein Mass-Modulated Effects in Alkaline Phosphatase

Abstract

Recent experimental studies engaging isotopically substituted protein (heavy protein) have revealed that many, but not all, enzymatic systems exhibit altered chemical steps in response to an altered mass. The results have been interpreted as femtosecond protein dynamics at the active site being linked (or not) to transition-state barrier crossing. An altered enzyme mass can influence several kinetic parameters (k cat , K m , and k chem ) in amounts of ≤30% relative to light enzymes. An early report on deuterium-labeled Escherichia coli alkaline phosphatase (AP) showed an unusually large enzyme kinetic isotope effect on k cat . We examined steady-state and chemical step properties of native AP, [ 2 H]AP, and [ 2 H, 13 C, 15 N]AP to characterize the role of heavy enzyme protein dynamics in reactions catalyzed by AP. Both [ 2 H]-and [ 2 H, 13 C, 15 N]APs showed unaltered steady-state and single-turnover rate constants. These findings characterize AP as one of the enzymes in which mass-dependent catalytic site dynamics is dominated by reactant-linked atomic motions. Two catalytic site zinc ions activate the oxygen nucleophiles in the catalytic site of AP. The mass of the zinc ions is unchanged in light and heavy APs. They are essentially linked to catalysis and provide a possible explanation for the loss of linkage between catalysis and protein mass in these enzymes.

Allosteric activation unveils protein-mass modulation of ATP phosphoribosyltransferase product release

“…The only other report of lack of heavy-enzyme isotope effects on the chemical step is the metalloenzyme alkaline phosphatase. In that instance, the unaltered mass of the catalytic Zn 2+ ions, involved in water activation and leaving group departure stabilisation, was invoked as a potential explanation for the decoupling between protein mass and chemical barrier crossing 23 . While ATPPRT is not per se a metalloenzyme, the reaction is dependent on Mg 2+ ions 26 , and crystal structures, computational chemistry calculations, and enzyme kinetics suggest it is involved directly in chemistry by positioning the substrates for nucleophilic attack and by stabilising the pyrophosphate leaving group 2 , 38 , 45 .…”

“…This was interpreted as evidence for coupling of fast protein dynamics to the chemical step, with a reduction in protein vibrational frequencies decreasing the probability of either crossing the transition-state energy barrier (purine nucleoside phosphorylase, HIV-1 protease, alanine racemase, lactate dehydrogenase) 5 , 6 , 18 , 20 or reaching the tunnelling-ready state (old yellow enzymes) 7 , 17 , or yet, increasing recrossing of the transition-state dividing surface (bacterial dihydrofolate reductase) 21 , 22 . As an exception, alkaline phosphatase showed no evidence for coupling of protein motions to chemistry 23 . Curiously, this approach has not yet been reported for enzymes modulated by allosteric effectors, even though protein motions at various timescales in these systems are proposed to mediate communication between allosteric effector binding and the active-site response 2 , 3 , 24 , 25 .…”

Capsulation of EBTAC into ZIF-8 for the development of a signal-on fluorescent biosensor to detect alkaline phosphatase

“…Fluorescence quantum yields were collected by a steady-state/lifetime spectrouorometer (Edinburgh FLS 1000, England). 1 H NMR and 13 C NMR spectra were acquired on a nuclear magnetic resonance spectrometer (Bruker Avance III 400 MHz, Germany). HRMS was recorded using a Thermo Scientic Q Exactive (America).…”

“…Alkaline phosphatase (ALP) is a glycoprotein that is widely distributed in biological tissues, which can hydrolyze phosphate esters into phosphoric acid in an alkaline environment. 1–3 As a key diagnostic biomarker, its abnormally high level usually means the occurrence of a certain disease, such as liver cancer, osteocarcinoma, leukemia, jaundice, skeletal illness and hypothyroidism. 4–7 Therefore, the development of a simple and convenient assay for the determination of ALP is urgent.…”