Comments on enzyme assays

Comments on enzyme assay

Amino Acid Racemase Enzyme Assays

Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide a detailed protocol using high performance liquid chromatography (HPLC) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, also Marfey’s reagent) for the characterization of novel amino acid racemases. The protocol described here was designed to obtain accurate kinetic parameters (kcat, KM values). Enzyme concentrations and reaction times were optimized so as to minimize the reverse reaction, which can confound results when measuring racemase reactions

Spectrophotometric Enzyme Assays for High-Throughput Screening

This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids

Fluorigenic enzyme assays using long-wavelength substrates

Investigations into the use of long-wavelength fluorigenic substrates, which are non- or weakly fluorescent but are converted by an appropriate enzyme to a highly fluorescent product, have been carried out in order to determine several different enzymes and their inhibitors. Certain nonfluorescent compounds based on long-wavelength xanthenes and oxazines dyes were synthesised. Amongst these, naphthofluorescein diphosphate and naphthofluorescein mono-phosphate has been demonstrated to be new fluorigenic substrates for alkaline phosphatase. The hydrolysis product of these substrates can be used for trace analyses using diode laser based fluorescence detection. [Continues.

Procedure for preparing permeabilized hyphae for enzyme assays

Procedure for preparing permeabilized hyphae for enzyme analysis

Assaying activity and assessing thermostability of hyperthermophilic enzymes

There is now a wide variety of intra- and extra-cellular enzymes available from organisms growing above 75°C, and having sufficient stability to allow assay well above this temperature. For some of these enzymes, to assay below even 95°C will involve measurement below the optimal growth temperature for the organism. The purpose of this chapter is to cover practical aspects of enzyme assay procedures that are specific to high temperatures. Since by far the commonest routine assessment of enzyme stability is activity loss, and because it is always unwise to measure enzyme activity without being confident of its stability during the assay, we include an outline of procedures for measuring enzyme activity loss/stability at high temperatures

Identifying Novel Inhibitors of RpFabG in Typhus-inducing Rickettsia prowazekii

Epidemic typhus is a rickettsial disease that is contracted via ticks and lice found on the flying squirrel. The disease is caused by Rickettsia prowazekii, an intracellular, gram-negative coccobacillus. The biosynthetic pathways of Rickettsia prowazekii and its host are intertwined. Therefore, the ideal antipathogenic drugs would not target a protein that is found within the biochemical pathway of the human host. There is a type II fatty acid synthase pathway that is unique to Rickettsia prowazekii, which can be distinguished from the multienzyme type I fatty- acid synthase pathway used in humans. Fab G 3-ketoacyl-(acyl-carrier-protein) reductase (RpFabG) is a protein that is specific to this type II pathway. Hence, this research is focused on finding a small molecule drug that inhibits RpFabG. The coding DNA sequence for the RpFabG protein was previously cloned into a pNICJBsa4 plasmid, which was transformed into BL21(DE3 Escherichia coli and Dh5α competent cells. The transformed bacteria were cultured in LB media, and the cells were harvested. The expressed protein was purified via Ni-NTA affinity chromatography, made possible by a His6 tag on the vector. Gel electrophoresis was performed to determine the purity of the obtained protein sample; due to indication of slight contamination, gel filtration fast protein liquid chromatography was run on a concentrated protein sample. Genetic Optimization of Ligand Docking (GOLD is a molecular docking software package that was used to rank potential inhibitors of RpFabG according to binding strength; ethyl acetoacetate (EAA and acetoacyl coenzyme A (AAC) were determined to have high binding strengths and thus determined to be strong potential inhibitors. An enzyme assay was run to determine the functionality of the enzyme, with EAA and AAC as substrates. AAC successfully decreased the enzymatic activity of RpFabG, suggesting its potential as a novel drug. DSF assay results were equivocal, indicating that inhibition assays should be run on AAC to further assess its potential as an inhibitor.College of Natural Science

Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed

Brush border digestion : development of a physiologically relevant in vitro model : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physiology at Massey University, Manawatū, New Zealand

Chapter Two has, in part, been published in the Springer publication, Reviews of Physiology, Biochemistry and Pharmacology, but the Author retains the right to publish the material in any collection consisting of the Author's own works: Hooton, D., Lentle, R., Monro, J., Wickham, M., & Simpson, R. (2015). The secretion and action of brush border enzymes in the mammalian small intestine. In: B. Nilius, T. Gudermann, R. Jahn, R. Lill, O. Petersen, & P. de Tombe (Eds.), Reviews of Physiology, Biochemistry and Pharmacology (vol 168, pp. 59-118). Cham: Springer. doi: 10.1007/112_2015_24The majority of current in vitro digestion methods either exclude the small intestinal brush border (BB) phase of digestion or do not incorporate the entire array of BB enzymes that are required to achieve terminal endogenous digestion in vivo. Accordingly, the digestate, and its derivitives, may not be representative of the digestive process in vivo. In order to improve the fidelity of the in vitro digestion process this thesis developed a physiologically relevant small intestinal BB phase using enzymes isolated from rat small intestinal mucosal tissue. The activities of BB enzymes were assessed and compared with known values, and under conditions physiologically representative of the small intestine. Although there were significant differences in BB enzyme activities depending on pH, enzyme solubilisation, and upon prolonged exposure to biliopancreatic secretions the BB preparation was deemed suitable for use in an in vitro digestion method. A rationale for the composition of the BB digestive phase was developed based on published physiological data, and was validated using glycosylated polyphenolic compounds as substrates. Liquid chromatography mass spectrometry (LC-MS) was used to assess the derivatisation products of BB digestion. In the absence of biliopancreatic secretions the onion flesh polyphenolic compounds quercetin-4ʹ-glucoside and isorhamnetin-4ʹ-glucoside, but not quercetin-3-glucoside or quercetin-34ʹ-diglucoside were hydrolysed. The positive control quercetin-3-glucoside was hydrolysed, and the negative control quercetin-3-rutinoside was not hydrolysed. The deglycosylation of quercetin-3-glucoside was monitored under conditions representative of the small intestine, i.e. incorporating bile and pancreatin, while at the appropriate pH. Quercetin-3-glucoside was significantly deglycosylated in BB treatments (no treatment or pancreatin alone) compared to BB treatments with bile (bile alone or pancreatin and bile). The mammalian digestive system is equipped to hydrolyse macronutrients from their polymeric form through to monomers and oligomers suitable for absorption across the epithelial layer. As such the inactivation or degradation of some BB enzymes during the BB digestive phase by bile or pancreatin was not unexpected, and does not preclude its use as an in vitro tool in the future