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A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK(a1)=4.9 and pK(a2)=7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K(m) values were obtained for different p-nitrophenyl beta-glycosides and K(i) values were determined for a range of alkyl beta-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E(187) (proton donor) and E(399) (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.

gamma-Cyclodextrin was found to be hydrolyzed by human salivary and pancreatic alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) at appreciable rates. The optimum pH for the enzyme reactions at 37 degrees C in the presence of 0.1 M NaCl was at around pH 5, which was remarkably different from the optimum pH (pH 6.9) of the enzymes for starch. The Km value (2.9 mg/ml) of pancreatic alpha-amylase for gamma-cyclodextrin was smaller than that (5.3 mg/ml) of salivary alpha-amylase at pH 5.3, while the V value of the former was 3.7-times larger than that of the latter. The hydrolyses of gamma-cyclodextrin by both enzymes took place via the multiple attack mechanism. The degrees of multiple attack by salivary and pancreatic alpha-amylases for gamma-cyclodextrin at pH 5.3 were 2.0 and 1.1, respectively. The distribution of maltodextrins produced by hydrolysis of gamma-cyclodextrin by salivary alpha-amylase was suggested to be independent of the substrate concentration, while that produced by pancreatic alpha-amylase was presumably dependent on the substrate concentration.

Maltohexaose producing amylase (EC 3.2.1.-) is the fourth known exo-amylase, the three previously known being glucoamylase, beta-amylase and Pseudomonas stutzeri maltotetraose producing amylase. The enzyme after release from Aerobacter aerogenes cells by 0.1% sodium lauryl sulfate extraction was purified by ammonium sulfate precipitation, DEAE-Sephadex column chromatography and Sephadex G-100 gel filtration to 80-fold of the original sodium lauryl sulfate extract activity, It gave a single band on disc electrophoresis, and the molecular weight by gel filtration was 54 000. This amylase showed maximal activity at 50 degrees C and pH 6.80. The pH stability range was relatively wide, the enzyme retaining more than 90% of its initial activity in the range of 6.50-9.0. 80% of the activity was retained after 15 min at 50 degrees C. This enzyme produced maltohexaose from starch, amylose and amylopectin by exo-attack, but did not act on alpha- or beta-cyclodextrin, pullulan or maltohexaitol. Also the enzyme acted on beta-limit dextrins of amylopectin and glycogen to form branched oligosaccharides. The unusual reaction of this enzyme on beta-limit dextrin is discussed from the standpoint of the stereochemistry of 1,4-alpha- and 1,6-alpha-glucosidic bonds. This is the anomalous amylase for which it is recognized that 1,6-alpha-glucosidic linkages in the substrates can mimic the effect of 1,4-alpha-bonds, as previously observed in pseudo-priming reactions of E. coli phosphorylase.

The 4,6,8(14)-triene-3-one steroids, highly fluorescent in aqueous solutions, lose their fluorescence power when binding occurs to hydrophobic regions of other molecules, such as the hydrophobic cavity in the ring system of cyclodextrins. The fluorescence intensity decreases almost completely when beta- and gamma-cyclodextrins are present in the solution. Scatchard plots derived from fluorescence titrations show that one or two molecules of steroid bind to one cyclodextrin molecule with KD,F-values of about 10(-4)-10(-5) mol/liter. Temperature-jump experiments show a single relaxation process, with rate constants for the decay of the beta-cyclodextrin-steroid complexes of about 10(4)-10(5) per s. For alpha- and gamma-cyclodextrins such relaxation processes are not observed.