No abstract

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

The kinetic parameters for the interconverting substrates D-xylose in equilibrium D-xylulose and D-glucose in equilibrium D-fructose were determined for several D-xylose isomerases, with Mg2+, Co2+ and Mn2+ as metal ion activators. The Km, kcat. and kcat./Km values are tabulated for the anomeric mixtures (observed parameters) as well as for the respective reactive species, i.e. the alpha-pyranose anomers of D-xylose and D-glucose and the alpha-furanose forms of D-xylulose and D-fructose (real parameters). The real Km values and catalytic efficiencies are more favourable for the ketose sugars (reverse reaction) than for the aldose sugars (forward reaction). Comparisons of the kinetic parameters further support the existence of two distinct groups of D-xylose isomerases. Inhibition constants for the cyclic substrate analogues 5-thio-alpha-D-xylopyranose and alpha-D-xylopyranosyl fluoride and for the acyclic substrate analogue xylitol and its dehydrated form 1,5-anhydroxylitol were determined and are discussed.

A highly specific 8-D-xylosidase with absolute glycon-substrate specificity was induced in Bacillus pumilus 12 by xylose. It was purified 34-fold by a procedure involving lysis of the cells with lysozyme, ammonium sulfate fractionation, G-75 Sephadex gel filtration and hydroxylapatite chromatography. The optimal activity was in the pH region 7.0-7.3. The isoelectric point was found to be 4.4. I n contrast to its broad complexing affinity, the hydrolytic activity was restricted to aryl @-D-xylopyranosides and lower xylo-oligosaccharide derivatives. From the dependence of K , and V,,, on pH, it could be deduced that two dissociable groups were necessary for catalytic activity. The enzyme was competitively inhibited by Tris at pH 7.2 and proved sensitive to ionic environment. p-Chloromercuribenzoate and other SH-reagents were inhbitory but substrate analogs and competitive inhibitors protected the enzyme against inactivation by p-chloromercuribenzoate. The results indicate that a histidine residue or imidazolium group and a sulfhydryl group might participate in the enzymatic hydrolysis of p-nitrophenyl 8-D-xylopyranoside. It has been reported that plants [l-61 and fungi [7-111 produce 8-D-xylosidases which are distinct from @-D-ghcosidases.A @-D-xylosidase from a commercial mold hemicellulase sample has been extensively studied in this laboratory reported the production of a pentosanase in Bacillus pumilus 12. This paper describes the induction, partial purification and some properties of a highly specific @-Dxylosidase in the same strain. The results were compared with those obtained for the fungal @-D-xY~o-sidase. Methyl B-D-lyxopyranoside was a gift from Dr. EXPERIMENTAL PROCEDURE Materials Kjolberg (Oslo).Pentoses, hexoses and xylitol were commercial products (Fluka, Merck). Lysozyme, dithiothreitol and Biogel P-150 were purchased from Calbiochem. ; deoxyribonuclease I from Worthington and streptomycin sulfate from Pfizer. Hydroxylapatite was prepared according to the method of Siegelman et al. [28]. Sephadex G-25 and G-75 were obtained from Pharmacia (Uppsala). Beef extract, yeast extract, agar and peptone were Oxoid products. All buffer materials and other reagents were analytical grade. MethodsRoutine enzymic assays were performed with p-nitrophenyl @-D-xylopyranoside as substrate a t a concentration of 4 mM in 0.05 M phosphate buffer, pH 7.2. The hydrolysis was followed by measuring the increase in absorbance a t 400 nm in a Beckman DB-G spectrophotometer. The sample compartment was thermostated a t 25" and the spectrophotometer was equipped with a linear-log Ten-Inch recorder.pa-Nitrophenyl 8-D-xylopyranoside hydrolysis as a function of pH was followed discontinuously by adding equal volumes of a 15 o/o Na2C0, solution to the reaction mixture [lo].Hydrolysis of phenyl and alkyl @-n-xylopyranosides was followed by measuring respectively phenol a t 280 nm [29] and liberated reducing sugar [30--311

Bacillus pumilus beta-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of beta-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with alpha- and beta-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of beta-D-xylopyranosyl fluoride at a high rate, V = 6.25 mumol min-1 mg-1 at 0 degrees C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon alpha-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, 1H NMR spectra of a digest of alpha-D-xylosyl fluoride showed the substrate to be specifically converted to alpha-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this "inverting" enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from alpha-D-xylosyl fluoride to form a beta-D-xylosidic product, followed by hydrolysis of the latter to produce alpha-D-xylose. The transient intermediate product formed enzymically from alpha-D-xylosyl fluoride in the presence of [14C]xylose was isolated and shown by its specific radioactivity and 1H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-beta-D-xylopyranosyl-D-xylopyranose containing one [14C]xylose residue.(ABSTRACT TRUNCATED AT 250 WORDS)