Elizabeth Ann Eigner

The specificity of enzymic reactions aminoacyl-soluble RNA ligases

Abstract 1. 1. Using the valine- and isoleucine-activating enzymes of Escherichia coli ( l -valine:sRNA ligase (AMP), EC 6.1.1.9 and l -isoleucine:sRNA ligase (AMP), EC 6.1.1.5, respectively), K m , K i and V have been determined for a variety of amino acid substrates under different conditions using pyrophosphate exchange, hydroxamate formation and sRNA acylation. 2. 2. By compairing the K m and V obtained under different assay conditions it has been possible to calculate K eq . (= k 2 / k 1 ) for the dissociation of the several enzyme-substrate complexes. 3. 3. The variation of K eq . with substrate structure is far greater than has been proposed on the basis of London forces, movement of the organic side chain into a “micro-organic phase on the enzyme surface” or specific side chain hydrophobic bonds. Space-filling considerations may be able to account for the differences in binding forces. It is probably necessary to invoke differences in the energy of the initial state conformation to account for the difference in binding of α-aminobutyric acid and valine. 4. 4. These changes in K eq . (as much as a 300-fold increase with the detection of one methylene group), together with small changes in V , suggest a relatively rigid structur for these enzymes and the participation of relatively strong bonds such as hydrogen bonds in enzyme-substrate fit. 5. 5. Unlike the carefully studied cases of chymotrypsin and certain esterases, there appears to be no correlation between substrate-enzyme binding and maximal reaction velocity. It follows that the substrate does, not, by its good fit to the enzyme, induce conformational changes which determine enzymic activity in the case of these enzymes.

A radioactive hydroxamate method for determining rates of amino acid activation

Abstract 1. 1. Amino acid-activating enzymes which catalyze the reaction between α-amino acids and ATP to form aminoacyl adenylates can be assayed by colorimetric measurement of the rate of formation of the corresponding hydroxamates in the presence of hydroxylamine or by measurement of the incorporation of radioactive pyrophosphate into ATP. The first of these techniques is quite insensitive and rarely used for quantitative work. The second technique is reliable and sensitive but cannot be used if more than one substrate is being activated or if there are other reactions occurring which lead to incorporation of pyrophosphate into ATP. 2. 2. By “activating” [ 14 C]amino acids in the presence of hydroxylamine it is possible to measure the formation of [ 14 C]amino acid hydroxamate. By assaying for the amount of [ 14 C]hydroxamate formed one is able to measure the extent of activation of one particular substrate, the 14 C-labeled substrate, regardless of whether other substrates are being activated or whether there are other pyrophosphate-incorporating reactions. 3. 3. Experimental details are provided which permit rapid and simple separation of [ 14 C]amino acid from [ 14 C]hydroxamate on ion-exchange paper, accurate assay of relative radioactivities and accurate estimation of reaction rates. The techniques are shown to be useful both with purified enzymes and crude tissue extracts.

[48] Kinetic techniques for the investigation of amino acid: tRNA ligases (aminoacyl-tRNA synthetases, amino acid activating enzymes)☆

Publisher Summary This chapter describes four kinetic assays for investigating amino acid:tRNA ligases: adenosine triphosphate:pyrophosphate (ATP:PPi) exchange assay, the hydroxamate assay, tRNA esterification assay, and adenosine monophosphate (AMP) production assay. The ATP:PPI exchange assay has been widely and profitably used to measure Kis for a variety of amino acid derivatives or ATP analogs that do not catalyze the ATP:PPI exchange. The hydroxamate technique is useful for the reaction of all the neutral amino acids. tRNA esterification method has been modified slightly to give best conditions for doing kinetic studies on purified ligases. This assay has the advantage of being very specific for a particular enzyme, amino acid, and tRNA. AMP production assay is responsive to any ATPases and to all kinds of ATP (AMP) ligases. In addition to the kinetic methods, some other techniques that have been especially useful in studying the kinetic properties of these enzymes are (1) Single-pass kinetics, (2) kinetic determinations of the isolated step of association and the membrane filter technique, (3) equilibrium and nonequilibrium dialysis techniques, and (4) possibly the most powerful tool, fluorescence analysis.

The preparation of pure [1-14C]- and [1-3H]-labeled l-amino acids☆

Abstract 1. 1. The preparation of high specific activity (30 mC/mmole) [1-14C]glycine, l -[1-14C]alanine, l -amino[1-14C]butyric acid, l -[1-14C]valine, l -[1-14C]alloisoleucine, l -[1-14C]isoleucine, l -[1-14C]norvaline, l -[1-14C]leucine, l -[1-14C]phenylalanine, l -[1-14C]tyrosine and l -p- fluoro [1- 14 C ] phenylalanine are described. 2. 2. l -[2,3-3H2]valine (1 C/mmole) and l -[2,3-3H2]isoleucine and -alloisoleucine have also been prepared by a unique route involving the catalytic addition of tritium gas to the corresponding oxazolones. 3. 3. Optically active 3-methylbutyraldehyde suffers very little racemization in its conversion to isoleucine in the Bucherer hydantoin synthesis. 4. 4. Extremely sensitive methods are described for determining chemical and optical purity of the labeled amino acids.