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.