A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity. Sugar-binding and crystallographic studies

The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments. Arabinose is bound and completely sequestered within the deep cleft between the two domains. With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction). Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP. To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis. Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose. The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged. We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars. Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding. Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein.     

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.     

Ligand recognition by the lactose permease of Escherichia coli: specificity and affinity are defined by distinct structural elements of galactopyranosides

Specificity of substrate recognition in lactose permease is directed toward the galactosyl moiety of lactose. In this study, binding of 31 structural analogues of D-galactose was examined by site-directed N-[(14)C]ethylmaleimide-labeling of the substrate-protectable Cys148 in the binding site. Alkylation of Cys148 is blocked by D-galactose with an apparent affinity of approximately 30 mM. Epimers of D-galactose at C-3 (D-gulose) and C-4 (D-glucose) or deoxy derivatives at these positions exhibit no binding whatsoever, indicating that these OH groups participate in essential interactions. Interestingly, the C-2 epimer alpha-D-talose binds almost as well as D-galactose, while 2-deoxy-D-galactose affords no substrate protection, indicating that nonstereospecific H-bonding at C-2 is required for stable binding. No substrate protection is detected with D-fucose, L-arabinose, 6-deoxy-6-fluoro-D-galactose, 6-O-methyl-D-galactose, or D-galacturonic acid, suggesting that the C-6 OH is an essential H-bond donor. Both alpha- and beta-methyl D-galactopyranosides bind more strongly than galactose, supporting the notion that the cyclic pyranose conformation is the bound form and that the anomeric configuration at C-1 does not contribute to substrate specificity. However, methyl or allyl alpha-D-galactopyranosides exhibit 60-fold lower apparent K(d)'s than D-galactose, demonstrating that binding affinity is significantly influenced by the functional group at C-1 and its orientation. Taken together, the observations confirm and extend the current binding site model [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] and indicate that specificity toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by hydrophobic interactions with the nongalactosyl moiety.     

Characterization of the mmsAB-araD1 (gguABC) genes of Agrobacterium tumefaciens

The chvE-gguABC operon plays a critical role in both virulence and sugar utilization through the activities of the periplasmic ChvE protein, which binds to a variety of sugars. The roles of the GguA, GguB, and GguC are not known. While GguA and GguB are homologous to bacterial ABC transporters, earlier genetic analysis indicated that they were not necessary for utilization of sugars as the sole carbon source. To further examine this issue, in-frame deletions were constructed separately for each of the three genes. Our growth analysis clearly indicated that GguA and GguB play a role in sugar utilization and strongly suggests that GguAB constitute an ABC transporter with a wide range of substrates, including L-arabinose, D-fucose, D-galactose, D-glucose, and D-xylose. Site-directed mutagenesis showed that a Walker A motif was vital to the function of GguA. We therefore propose renaming gguAB as mmsAB, for multiple monosaccharide transport. A gguC deletion affected growth only on L-arabinose medium, suggesting that gguC encodes an enzyme specific to L-arabinose metabolism, and this gene was renamed araD1. Results from bioinformatics and experimental analyses indicate that Agrobacterium tumefaciens uses a pathway involving nonphosphorylated intermediates to catabolize L-arabinose via an L-arabinose dehydrogenase, AraA(At), encoded at the Atu1113 locus.     

Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the structure and function of this regulatory protein.     

Pentose metabolism in Mycobacterium smegmatis: specificity of induction of pentose isomerases.

The induction of D-xylose, D-ribose, L-arabinose, and D-lyxose isomerases by various sugars was studied to determine the configuration necessary for induction. D-Xylose isomerase was only induced by D-xylose, whereas D-ribose isomerase was induced by D-ribose, L-rhamnose, and L-lyxose. L-arabinose isomerase was induced by L-arabinose, D-galactose, L-arabitol, D-fucose, and dulcitol, whereas D-lyxose isomerase was induced by D-lyxose, D-mannose, D-ribose, dulcitol, and myoinositol. Some compounds such as dulcitol, D-galactose, and D- or L-fucose which do not support growth are still able to serve as inducers for various pentose isomerases.     

Characteristics of D-galactose transport systems by luminal membrane vesicles from rabbit kidney

The characteristics of renal transport of D-galactose by luminal membrane vesicles from either whole cortex, pars recta or pars convoluta of rabbit proximal tubule were investigated by a spectrophotometric method using a potential-sensitive carbocyanine dye. Uptake of D-galactose by luminal membrane vesicles prepared from whole cortex was carried out by an Na+-dependent and electrogenic process. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for the uptake of D-galactose. Tubular localization of the transport systems was studied by the use of vesicles derived from pars recta and pars convoluta. In pars recta, Na+-dependent transport of D-galactose and D-glucose occurred by means of a high-affinity system (half-saturation: D-galactose, 0.15 +/- 0.02 mM; D-glucose, 0.13 +/- 0.02 mM). These results indicated that the "carrier' responsible for the uptake of these hexoses does not discriminate between the steric position of the C-4 hydroxyl group of these two isomers. This is further confirmed by competition experiments, which showed that D-galactose and D-glucose are taken up by the same and equal affinity transport system by these vesicle preparations. Uptake of D-galactose and D-glucose by luminal membrane vesicles isolated from pars convoluta was mediated by a low-affinity common transport system (half-saturation: D-galactose, 15 +/- 2 mM; D-glucose, 2.5 +/- 0.5 mM). These findings strongly suggested that the "carrier' involved in the transport of monosaccharides in vesicles from pars convoluta is specific for the steric position of the C-4 hydroxyl group of these sugars and presumably interacts only with D-glucose at normal physiological concentration.     

Structural requirements for binding to the sugar-transport system of the human erythrocyte

The structural requirements for binding to the glucose/sorbose-transport system in the human erythrocyte were explored by measuring the inhibition constants, K(i), for specifically substituted analogues of d-glucose when l-sorbose was the penetrating sugar. Derivatives in which a hydroxyl group in the d-gluco configuration was inverted, or replaced by a hydrogen atom, at C-1, C-2, C-3, C-4 or C-6 of the d-glucose molecule, all bound to the carrier, confirming that no single hydroxyl group is essential for binding to the carrier. The binding and transport of 1-deoxy-d-glucose confirmed that the sugars bind in the pyranose form. The relative inhibition constants of d-glucose and its deoxy, epimeric and fluorinated analogues are consistent with the combination of beta-d-glucopyranose with the carrier by hydrogen bonds at C-1, C-3, probably C-4, and possibly C-6 of the sugar. Both polar and non-polar substituents at C-6 enhance the affinity of d-glucose derivatives relative to d-xylose, and d-galactose derivatives relative to l-arabinose, and it is suggested that the carrier region around C-6 of the sugar may contain both hydrophobic and polar binding groups. The spatial requirements at C-1, C-2, C-3, C-4 and C-6 were explored by comparing the relative binding of d-glucose and its halogeno and O-alkyl substituents. The carrier protein closely approaches the sugar except at C-3 in the d-gluco configuration, C-4 and C-6. d-Glucal was a good inhibitor, showing that a strict chair form is not essential for binding. 3-O-(2',3'-Epoxypropyl)-d-glucose, a potential substrate-directed alkylating agent, bound to the carrier, but did not inactivate it.     

Induction of NADPH-linked D-xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D-xylose, L-arabinose, or D-galactose

Considerable interest in the D-xylose catabolic pathway of Pachysolen tannophilus has arisen from the discovery that this yeast is capable of fermenting D-xylose to ethanol. In this organism D-xylose appears to be catabolized through xylitol to D-xylulose. NADPH-linked D-xylose reductase is primarily responsible for the conversion of D-xylose to xylitol, while NAD-linked xylitol dehydrogenase is primarily responsible for the subsequent conversion of xylitol to D-xylulose. Both enzyme activities are readily detectable in cell-free extracts of P. tannophilus grown in medium containing D-xylose, L-arabinose, or D-galactose and appear to be inducible since extracts prepared from cells growth in media containing other carbon sources have only negligible activities, if any. Like D-xylose, L-arabinose and D-galactose were found to serve as substrates for NADPH-linked reactions in extracts of cells grown in medium containing D-xylose, L-arabinose, or D-galactose. These L-arabinose and D-galactose NADPH-linked activities also appear to be inducible, since only minor activity with L-arabinose and no activity with D-galactose is detected in extracts of cells grown in D-glucose medium. The NADPH-linked activities obtained with these three sugars may result from the actions of distinctly different enzymes or from a single aldose reductase acting on different substrates. High-performance liquid chromatography and gas-liquid chromatography of in vitro D-xylose, L-arabinose, and D-galactose NADPH-linked reactions confirmed xylitol, L-arabitol, and galactitol as the respective conversion products of these sugars. Unlike xylitol, however, neither L-arabitol nor galactitol would support comparable NAD-linked reaction(s) in cellfree extracts of induced P. tannophilus. Thus, the metabolic pathway of D-xylose diverges from those of L-arabinose or D-galactose following formation of the pentitol.     

Comparison of the periplasmic receptors for L-arabinose, D-glucose/D-galactose, and D-ribose. Structural and Functional Similarity.

The primary sequence of the receptor for L-arabinose or Ara-binding protein (ABP) composed of 306 residues is very different from the D-glucose/D-galactose-binding protein (GGBP) which consists of 309 residues. Nevertheless, superimpositioning of the well-refined high resolution structures of ABP in complex with D-galactose and the GGBP in complex with D-glucose shows very similar structures; 220 of the residues (or about 70%) have a root mean square deviation of 2.0 A. From the superpositioning, nine pairs of continuous segments (consisting of 8-51 residues), mainly alpha-helices and beta-strands that form the core of the two lobes of the bilobate proteins were found to exhibit strong sequence homology. The equivalenced structures and aligned sequences show that many of the polar, as well as aromatic residues, in the sugar-binding sites located in the cleft between the two lobes are highly conserved. Surprisingly, however, the exact mode of binding of the D-galactose in ABP is totally different from that of the D-glucose in GGBP. Using the structurally aligned sequences of the ABP and GGBP as a template, we have matched the sequence of the ribose-binding protein (RBP) which consists of 271 residues with the ABP/GGBP pair. Although the nine aligned segments of all three proteins show little sequence identity, they have significant homology. Four additional segments of RBP were matched only with GGBP, leading to the alignment of about 90% of the RBP sequence with the GGBP sequence. Many of the conserved residues in the binding sites of ABP and GGBP matched with similar residues in RBP. Additional observations indicate that the GGBP/RBP pair is more closely related than the ABP/RBP or ABP/GGBP pair. All three binding proteins, which may have diverged from a common ancestor, serve as primary receptors for bacterial high affinity active transport systems. Moreover, GGBP and RBP, but not ABP, also act as receptors for chemotaxis. An exposed site located in one domain, which includes Gly74, for interacting with the trg transmembrane signal transducer that is involved in triggering chemotaxis has been located in the structure of GGBP (Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (1988) Science 242, 1290-1295). Whereas the site is absent in the structure of ABP, it is strongly predicted to be present in RBP which shares the same trg transducer with GGBP. The knowledge-based alignment of RBP further revealed two possible additional peripheral chemotactic sites that show high structural and sequence similarity between GGBP and RBP only. At least one of these sites, together with the one proven to exist in the other domain, could be used by the signal transducer with which both binding proteins interact in a way which the substrate-loaded "closed cleft" structure could be discriminated from the unliganded "open cleft" form by the transducer.     

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen

The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.     

Competitive kinetics of sugar active transport in snail intestine.

Everted intestinal rings of the snail Cryptomphalus hortensis accumulate labelled sugars against a concentration gradient. The active transport of D-galactose (KT = 3.6 mM) is competitively inhibited by D-glucose (Ki = 8.2 mM) and by 3-0-methylglucose (Ki = 24 mM), but it is not affected by L-arabinose, D-fructose, L-arabinose and D-mannitol penetrate into the tissue at the same rate, they do not develop accumulation gradient, and all of them follow the kinetics of a diffusion process. D-glucose, on the contrary, like galactose, penetrates much more quickly and accumulates against a gradient.     

The catalytic mechanism of galactose mutarotase

Galactose mutarotase catalyzes the first step in normal galactose metabolism by catalyzing the conversion of beta-D-galactose to alpha-D-galactose. The structure of the enzyme from Lactococcus lactis was recently solved in this laboratory and shown to be topologically similar to domain 5 of beta-galactosidase. From this initial X-ray analysis, four amino acid residues were demonstrated to be intimately involved in sugar binding to the protein: His 96, His 170, Asp 243, and Glu 304. Here we present a combined X-ray crystallographic and kinetic analysis designed to examine the role of these residues in the reaction mechanism of the enzyme. For this investigation, the following site-directed mutant proteins were prepared: H96N, H170N, D243N, D243A, E304Q, and E304A. All of the structures of these proteins, complexed with either glucose or galactose, were solved to a nominal resolution of 1.95 A or better, and their kinetic parameters were measured against D-galactose, D-glucose, L-arabinose, or D-xylose. From these studies, it can be concluded that Glu 304 and His 170 are critical for catalysis and that His 96 and Asp 243 are important for proper substrate positioning within the active site. Specifically, Glu 304 serves as the active site base to initiate the reaction by removing the proton from the C-1 hydroxyl group of the sugar substrate and His 170 functions as the active site acid to protonate the C-5 ring oxygen.     

Molecular dynamics simulations of the auxin‐binding protein 1 in complex with indole‐3‐acetic acid and naphthalen‐1‐acetic acid

Auxin‐binding protein 1 (ABP1) is suggested to be an auxin receptor which plays an important role in several processes in green plants. Maize ABP1 was simulated with the natural auxin indole‐3‐acetic acid (IAA) and the synthetic analog naphthalen‐1‐acetic acid (NAA), to elucidate the role of the KDEL sequence and the helix at the C‐terminus. The KDEL sequence weakens the intermolecular interactions between the monomers but stabilizes the C‐terminal helix. Conformational changes at the C‐terminus occur within the KDEL sequence and are influenced by the binding of the simulated ligands. This observation helps to explain experimental findings on ABP1 interactions with antibodies that are modulated by the presence of auxin, and supports the hypothesis that ABP1 acts as an auxin receptor. Stable hydrogen bonds between the monomers are formed between Glu40 and Glu62, Arg10 and Thr97, Lys39, and Glu62 in all simulations. The amino acids Ile22, Leu25, Trp44, Pro55, Ile130, and Phe149 are located in the binding pocket and are involved in hydrophobic interactions with the ring system of the ligand. Trp151 is stably involved in a face to end interaction with the ligand. The calculated free energy of binding using the linear interaction energy approach showed a higher binding affinity for NAA as compared to IAA. Our simulations confirm the asymmetric behavior of the two monomers, the stronger interaction of NAA than IAA and offers insight into the possible mechanism of ABP1 as an auxin receptor. Proteins 2014; 82:2744–2755. © 2014 Wiley Periodicals, Inc.     

Intramolecular hydrogen bonds and conformation of the lincomycin molecule in organic solvents

IR spectra (1600-1800 and 3000-3650 cm-1) of lincomycin base solutions in inert (CCl4 and C2Cl4), proton acceptor (dioxane, dimethylsulfoxide and triethyl amine) and proton donor (CHCl3, CD3OD and D2O) solvents were studied. Analysis of the concentration and temperature changes in the spectra revealed that association in lincomycin in the inert solvents was due to intramolecular hydrogen linkage involving amide and hydroxyl groups. Disintegration of the associates after the solution dilution and temperature rise was accompanied by formation of intramolecular bonds stabilizing the stable conformation structure of the lincomycin molecule. The following hydrogen linkage in the conformation was realized: NH...N (band v NH...N at 3340 cm-1), OH...O involving the hydroxyl at C-7 and O atoms in the D-galactose ring (band v OH...O at 3548 cm-1), a chain of the hydrogen bonds OH...OH...OH in the lincomycin carbohydrate moiety (band v OH...O at 3593 cm-1 and v OH of the end hydroxyl group at 3625 cm-1). Bonds NH and C-O of the amide group were located in transconformation. Group C-O did not participate in the intramolecular hydrogen linkage.     

Conversion of sugars to 1,2-propanediol by Thermoanaerobacterium thermosaccharolyticum HG-8

The purpose of this study was to explore a fermentation route for the production of 1,2-propanediol (1,2-PD) from renewable sugars: lactose found in cheese whey, and D-glucose, D-galactose, L-arabinose, and D-xylose found in corn and wood byproducts. Thermoanaerobacterium thermosaccharolyticum, a naturally occurring organism, was found to ferment a wider range of sugars to 1,2-PD than previously reported. The specific sugar had a significant effect on the selectivity for 1,2-PD vs other fermentation products such as ethanol, D- and L-lactate, and acetate. T. thermosaccharolyticum potentially provides an environmentally friendly route to a major commodity chemical now made from petrochemicals.     

The localization of a lectin-like component on the Leishmania cell surface

The binding between marcrophage-like cells J774G8 and Leishmania braziliensis (NR) promastigotes was studied in vitro by a radioisotopic assay under various conditions in the absence of serum. Different sugars, N-acetyl-D-glucosamine, D-glucose, D-mannose, D-galactose, and chitin, diminished the binding of the parasite, whereas other sugars, D-arabinose, D-fucose and D-xylose, did not affect the binding. The presence of a lectin-like ligand specific for N-acetyl-D-glucosamine has been detected on the cell surface of the Leishmania braziliensis (NR) by fluorescence microscopy.These data suggest that the binding of the parasite to the host's cell is a ligand-receptor interaction which involves the participation of a lectin-like component on the parasite cell surface.     

The interaction of Ricinus communis hemagglutinin with polysaccharides and low molecular weight carbohydrates

The hemagglutinin from the castor bean (Ricinus communis) shows a precipitin-like like reaction with a series of branched galactomannas, dependent on their galactose: mannose ratio. Charged and neutral linear galactants fail to co-precipitate with the protein. Hapten inhibition of the turbidimetrically assayed hemagglutinin-Lucerne seed galactomannan system incidates that simple sugars such as D-galactose, D-fucose and L-arabinose bind to the protein. Of the glycosides tested, methyl β-D-galactopyranoside is a better inhibitor than the corresponding α-another. $\text{p-}\text{Nitrophenyl}\text{-2-}\text{acetamido}\text{-2-}\text{deoxy}\text{-β-}\text{D}\text{-}\text{galactopyranoside}$ is about 10 tiems less effective than $\text{p-}\text{nitrophenyl}\text{-β-}\text{D}\text{-}\text{galactopyranoside}$, the best inhibitor tested. Equilibrium dialysis data obatined with the latter ligand are consistent with a protein containing two identical and independent binding sites with an intrinsic association constant equal to 1.65 ? 10⁴ l/mole at 25 °C.