The crystal structures of family 10 xylanases indicate that the distal regions of their active sites are quite different, suggesting that the topology of the substrate binding clefts of these enzymes may vary. To test this hypothesis, we have investigated the rate and pattern of xylooligosaccharide cleavage by the family 10 enzymes, Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) and Cellulomonas fimi exoglucanase, Cex. The data showed that Cex contained three glycone and two aglycone binding sites, while XYLA had three glycone and four aglycone binding sites, supporting the view that the topologies of substrate binding clefts in family 10 glycanases are not highly conserved. The importance of residues in the substrate binding cleft of XYLA in catalysis and ligand binding were evaluated using sitedirected mutagenesis. In addition to providing insight into the function of residues in the glycone region of the active site, the data showed that the aromatic residues Phe-181, Tyr-255, and Tyr-220 play important roles in binding xylose moieties, via hydrophobic interactions, at subsites ؉1, ؉3, and ؉4, respectively. Interestingly, the F181A mutation caused a much larger reduction in the activity of the enzyme against xylooligosaccharides compared with xylan. These data, in conjunction with a previous study (Charnock, S. J., Lakey, J. H., Virden, R., Hughes, N., Sinnott, M. L., Hazlewood, G. P., Pickersgill, R., and Gilbert, H. J. (1997) J. Biol. Chem. 272, 2942-2951), suggest that the binding of xylooligosaccharides at the ؊2 and ؉1 subsites ensures that the substrates occupy the ؊1 and ؉1 subsites and thus preferentially form productive complexes with the enzyme. Loss of ligand binding at either subsite results in small substrates forming nonproductive complexes with XYLA by binding to distal regions of the substrate binding cleft.

In a previous study crystals of Pseudomonas fluorescens subspecies cellulosa xylanase A (XYLA) containing xylopentaose revealed that the terminal nonreducing end glycosidic bond of the oligosaccharide was adjacent to the catalytic residues of the enzyme, suggesting that the xylanase may have an exo-mode of action. However, a cluster of conserved residues in the substrate binding cleft indicated the presence of an additional subsite, designated subsite F. Analysis of the biochemical properties of XYLA revealed that the enzyme was a typical endo-␤1,4-xylanase, providing support for the existence of subsite F. The three-dimensional structure of four family 10 xylanases, including XYLA, revealed several highly conserved residues that are on the surface of the active site cleft. To investigate the role of some of these residues, appropriate mutations of XYLA were constructed, and the biochemical properties of the mutated enzymes were evaluated. N182A hydrolyzed xylotetraose to approximately equal molar quantities of xylotriose, xylobiose, and xylose, while native XYLA cleaved the substrate to primarily xylobiose. These data suggest that N182 is located at the C site of the enzyme. N126A and K47A were less active against xylan and aryl-␤-glycosides than native XYLA. The potential roles of Asn-126 and Lys-47 in the function of the catalytic residues are discussed. E43A and N44A, which are located in the F subsite of XYLA, retained full activity against xylan but were significantly less active than the native enzyme against oligosaccharides smaller than xyloseptaose. These data suggest that the primary role of the F subsite of XYLA is to prevent small oligosaccharides from forming nonproductive enzyme-substrate complexes.

Mannanase A (MANA) from Pseudomonas fluorescens, a member of glycosyl hydrolase family 26, was hyperexpressed in Escherichia coli and purified to homogeneity. Analysis of the stereochemical course of mannotetraose hydrolysis by purified MANA showed that the configuration of the anomeric carbon was retained on cleavage of the middle glycosidic bond. These data suggest that the mannanase hydrolyzes mannooligosaccharides by a double-displacement general acid-base mechanism. By hydrophobic cluster analysis (HCA), two glutamate and two aspartate residues were shown to be conserved in all of the glycosyl hydrolase family 26 enzymes analyzed. In addition, HCA suggested that family 26 was related to the GH-A clan (families 1, 2, 5, 10, 30, 35, 39, and 42) of (alpha/beta)8-barrel glycosyl hydrolases, which led to the prediction that E320 and E212 constitute the catalytic nucleophile and acid-base residues, respectively. To investigate the role of these amino acids, site-directed mutagenesis was used to replace the two aspartates with alanine and glutamate, while the two conserved glutamates were changed to alanine and aspartate. The mutant enzymes were purified and their biochemical properties were analyzed. The data showed that neither the D-->A nor the D-->E mutation resulted in a dramatic decrease in enzyme activity, suggesting that the two aspartate residues did not play a pivotal role in catalysis. In contrast, modification of either of the glutamate residues to alanine caused a dramatic decrease in kcat against carob galactomannan, azo-carob galactomannan, mannotetraose and 2,4-dinitrophenyl beta-mannobioside (2,4-DNPM). The E320A mutation did not alter the apparent K(m) (K(m)) of MANA against these substrates, while E212A resulted in a 27-fold decrease in K(m) against 2,4-DNPM. Pre-steady-state kinetics of 2,4-DNPM hydrolysis by E212A showed that there was a rapid burst of 2,4-dinitrophenol release. Circular dichroism and fluorescence spectroscopy indicated that there were no significant differences between the structures of the mutant and wild-type forms of MANA. These data are consistent with E212 and E320 constituting the catalytic acid-base and nucleophile residues of MANA, respectively.

1. A simple chromatographic method is described for the purification of arginine kinase from lobster (Homarus vulgaris) muscle. 2. Some physical properties and the effects on enzyme activity of ionic strength, pH, buffer salts, metal ions and substrates are reported. 3. The kinetic parameters, evaluated by variation of the concentration of one of the substrates, are dependent on the concentration of the other substrate. 4. The properties of the enzyme are discussed in relation to previous findings about phosphagen phosphotransferases.