Transport of Sugars and Amino Acids in Bacteria. XVII. On the Existence and Nature of Substrate Amino Acids Bound to Purified Branched Chain Amino Acid-binding Proteins of Escherichia coli1

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Abstract
The substrate binding reactions of purified, homogeneous leucine-isoleucine-valine-threonine binding protein (LIVT-binding protein) and leucine specific binding protein (Ls-binding protein) of Escherichia coli were extensively examined by an equilibrium dialysis method using radioactive substrates. The concave downward Lineweaver-Burk plots for the reactions were obtained and they showed that the maximal number of binding sites per molecule of each protein was 1. It was also found that the specific binding activity of LIVT-binding protein was significantly altered by the protein concentration. Dansylation with dansyl chloride and subsequent thin layer chromatography of the purified LIVT-binding protein revealed the existence of isoleucine, and leucine and/or valine, bound to the protein noncovalently. The total amount of these amino acids was about equimolar to that of the protein. These bound amino acids were found to be exchanged with an added substrate amino acid when the protein was dialyzed against buffer containing one of the substrate amino acids of this protein. Using the protein containing radioactive isoleucine as a bound substrate, the exchange reaction was shown to occur rapidly and stoichiometrically. Release of these bound amino acids from the protein solution was found to occur very slowly in the absence of the exchange reaction on dialysis or during gel filtration. The affinity of the bound amino acids for the protein was measured using protein depleted of the bound substrates by treatment with 6 M urea. Substrate binding reactions of the depleted protein showed Michaelis-Menten type kinetics with dissociation constants of 1–2×10−7 M and nearly 1 as the maximal number of binding sites. These findings indicated that the substrate binding reaction of LIVT-binding protein is a simple association-dissociation reaction with dissociation constants of around 1×10−7 M for its substrates, and that owing to these small dissociation constants the purified protein can retain about an equimolar amount of its substrates which are in a dynamic equilibrium with the protein. Based on these findings and theoretical considerations, a rationale explaining multiphasic binding kinetics produced by binding protein having a small dissociation constant for substrate and a single binding site in the molecule was presented. Similar conclusions were made about Ls-binding protein in which a small amount of leucine was detected by dansylation. Purification and properties of the third binding protein for branched chain amino acids of E. coli were described.