Efficient factors in protein modification: ADenosine deaminase esterification by woodward reagent K

Abstract

Chemical modification of Adenosine Deaminase (ADA) with N-ethyl-5-phenyl isoxazoliom-3 -sulfonate (Woodward's reagent K) (WR-K) was studied using experimental and theoretical techniques. Reaction concentration ranges were 0.8-6 mM WR-K at pH 7.8 and 27 °C. It was observed that the maximum number of moles of esterified residues per mol of enzyme ( ) in this concentration ranges is 4. However, esterification of ADA does not affect the activity of ADA, suggesting that the active site residues are not esterified. Similar results were obtained when the active site was blocked with 0.1 mM erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), followed by esterification, as measured by enol ester formation using absorbance at 340 nm. A theoretical approach was employed to study the modification process using molecular dynamic simulation, MM and QM/MM minimization. A full ASA empirical model and B3LYP method were used to evaluate the relative stability of some species which may arise from the reaction of ADA with WR-K. Theoretical results have shown that five residues (Glu 244, Glu 121, Glu 337, Asp 127, Asp 338) can be possible cases for modification in reaction 1:1 between ADA and WR-K at = 1. Glu 121 was possible initially modified in this process. Besides, it is specified that atomic accessible surface area cannot be an appropriate criterion in determination of primary sites which are modified by WR-K. Ultimately, it is clarified that among effective factors in modification of enzyme surface such as atomic accessible surface, stability of modified segment and local residues changes of ADA, latter factor plays a basic role in this process from kinetics and thermodynamics point of view.

Acidic residue modifications restore chaperone activity of β-casein interacting with lysozyme

“…The modification of the ␤-casein by WRK showed a new absorption peak in the wavelength range 340-350 nm, establishing the modification of aspartate and glutamate residues [33,48]. The first step in WRK reaction is the formation of ketoketenimine, which then reacts with carboxylate groups to produce an enol ester [49] with an absorbance peak at 340-350 nm. CD spectra of WRK-modified ␤-casein showed more negative ellipticity relative to native ␤-casein in the range of 250-204 nm (Fig.…”

Covalent Protein Labeling at Glutamic Acids

“…As such it is stable, but it can be activated by bases initiating a rearrangement to form a ketenimine that reacts with carboxylic acids to form enol esters. Due to this reactivity, WRK has also been employed in enzymology for analysis of enzyme mechanisms through covalent modification of key carboxylic acids and other amino acids (Bustos et al, 1996;Carvajal et al, 2004;Komissarov et al, 1995;Mahnam et al, 2008;Sinha and Brewer, 1985).…”

“…Woodward's reagent K (WRK) (Kemp and Chien, 1967;) ( Figure 1A) has been employed in protein chemistry to covalently modify nucleophilic cysteine (Bustos et al, 1996), histidine (Carvajal et al, 2004), and carboxylic acid residues (Bodlaender et al, 1969;Feinstein et al, 1969;Komissarov et al, 1995;Mahnam et al, 2008;Pé tra and Neurath, 1971;Pé tra, 1971;Sinha and Brewer, 1985) in order to study enzymatic activity. We have recently described that ABPP probes based on WRK can be surprisingly selective and covalently modify the N-terminal proline of macrophage migration inhibitory factor (Qian et al, 2016).…”