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Galactokinases (GalKs) have attracted significant research attention for their potential applications in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 presents a remarkably broad substrate range including 14 diverse natural and unnatural sugars. TLC and MS studies revealed that GalKSpe4 had relaxed activity towards galactose derivatives with modifications on the C-6, 4- or 2-positions. Additionally, GalKSpe4 can also tolerate glucose while glucose derivatives with modifications on the C-6, 4- or 2-positions were unacceptable. More interestingly, GalKSpe4 can phosphorylate l-mannose in moderate yield (43%), while other l-sugars such as l-Gal cannot be recognized by this enzyme. These results are very significant because there is rarely enzyme reported that can phosphorylate such uncommon substrates as l-mannose.

We report the thermodynamics of binding of D-galactose and deoxy derivatives thereof to the arabinose binding protein (ABP). The "intrinsic" (solute-solute) free energy of binding ΔG°int at 308 K for the 1-, 2-, 3-, and 6-hydroxyl groups of gal

A recently developed technique for the probing of the combining sites of lectins and antibodies, to establish the structure of the epitope that is involved in the binding of an oligosaccharide, is used to study the binding of methyl α-isomaltoside by the enzyme glucoamylase. The procedure involved the determination of the effects on the kinetics of hydrolysis of both monodeoxygenation and mono-O-methylation at each of the seven hydroxyl groups in order to gain an estimate of the differential changes in the free energies of activation, ΔΔG(paragraph). As expected, from previous publications, both deoxygenation and O-methylation of OH-4 (reducing unit), OH-4', or OH-6' strongly hindered hydrolysis, whereas the kinetics were virtually unaffected by either the substitutions at OH-2 or structural changes at C-1. The substitutions at OH-3 caused increases of 2.1 and 1.9 kcal/mol in the ΔΔG(paragraph). In contrast, whereas deoxygenation of either OH-2' or OH-3' caused much smaller (0.96 and 0.52 kcal/mol) increases in ΔΔG(paragraph), the mono-O-methylations resulted in severe steric hindrance to the formation of the activated complex. The relatively weak effects of deoxygenation suggest that the hydroxyl groups are replaced by water molecules and thereby participate in the binding by contributing effective complementarity. Methyl α-isomaltoside was docked into the combining site of the X-ray crystal structure at 2.4 A resolution of the complex with the inhibitor acarbose. A fit free of steric interactions with the protein was found that has the methyl α-glucopyranoside unit in the normal 4C1 conformation and the other glucose unit approaching a half-chair conformation with the interunit fragment defined by the torsion angles Φ/ψ/ω = 74°/134°/166° (O-5'-C-1'(φ)-O-6(ψ)-C-6(ω)-C-5-O-5). The model provides a network of hydrogen bonds that appears to well represent the activated complex formed by the glucoamylase with both maltose and isomaltose since the structures appear to provide a sound rationale for both the specificity and catalysis provided by the enzyme. A recently developed technique for the probing of the combining sites of lectins and antibodies, to establish the structure of the epitope that is involved in the binding of an oligosaccharide, is used to study the binding of methyl α-isomaltoside by the enzyme glucoamylase. The procedure involved the determination of the effects on the kinetics of hydrolysis of both monodeoxygenation and mono-O-methylation at each of the seven hydroxyl groups in order to gain an estimate of the differential changes in the free energies of activation. A model has been developed which provides a network of hydrogen bonds that appears to well represent the activated complex formed by the glucoamylase with both maltose and isomaltose because the structures appear to provide a sound rationale for both the specificity and catalysis provided by the enzyme.

Modified aldopentonic and aldohexonic acids were synthesized in order to study the electronic requirements for a successful enzymatic conversion into their corresponding 2-keto 3-deoxy analogues by dihydroxy acid dehydratase (DHAD), an enzyme from the biosynthetic pathway of branched chain amino acids. Analytical tests with the novel artificial substrates (18)-(21) and (27) provided evidence that the amount of conversion could be enhanced by replacement of the hydroxy group at C 4 of L-arabinonic acid (21) with less electron-withdrawing, ambivalent or electron-donating substituents. Modified aldohexonic acids were no substrates for DHAD, perhaps due to less perfect binding to the active site presumably for steric reasons. For 4-deoxy-L-threo-pentonic acid (18) the enzymatic conversion into 3,4-dideoxy-2-ketopentonic acid (29) by DHAD could be achieved on a preparative scale.

The syntheses of three deoxy-α-D-"glucopyranosyl" phosphates and a series of dideoxymonofluoro- and dideoxydifluoro-α-D-glucopyranosyl phosphates are described.Rate constants for their acid-catalyzed hydrolysis were determined.Deoxygenation in the sugar r

Syntheses are reported of 4-deoxy-D-xylo-hexose and 4-azido-4-deoxy-D-glucose as potential inhibitors for lactose synthase.These syntheses involved S

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