Wild-type and mutant D-xylose isomerase from Actinoplanes missouriensis: metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects.

The metal-ion dissociation constants (Mg2+, Mn2+) of wild-type and mutant D-xylose isomerases from Actinoplanes missouriensis have been determined by titrating the metal-ion-free enzymes with Mg2+ and Mn2+ respectively. Substitution of amino acids co-ordinated to metal-ion 1 (E181D, D245N) dramatically affects the dissociation constants, pH-activity profiles and apparent substrate binding. Mutagenesis of groups ligated to metal-ion 2 is less drastic except for that of Asp-255: a decrease in metal-ion affinity, a change in metal-ion preference and an improved apparent substrate binding (at pH values above the optimum), especially in the presence of Mn2+, are observed for the D255N enzyme. Similar effects, except for a slightly increased metal-ion affinity, are obtained by mutagenesis of the adjacent Glu-186 to Gln and the unconserved Ala-25 to Lys. Moreover, the striking acidic-pH shifts observed for the D255N and E186Q enzymes support the crucial role of the water molecule, Wa-690, Asp-255 and the adjacent Glu-186 in proton transfer from 2-OH to O-1 of the open and extended aldose substrate. Mutations of other important groups scarcely affect the metal-ion dissociation constants and pH-activity profiles, although pronounced effects on the kinetic parameters may be observed.

Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases.

D-Xylose isomerases are metal-ion (Mn2+, Co2+, Mg2+)-requiring tetrameric enzymes. Both the stoichiometry and the binding constants have been determined by titrating the metal-ion-free enzymes from five organisms (Actinomycetaceae and more divergent bacteria) with the respective metal ions using the enzyme activity as indicator of active complex-formation. The following characteristics have been observed for each specific isomerase: (i) two essential metal ion sites (one structural and one catalytic) exist per subunit; (ii) the metal ion binding at one site does not affect the binding at the other site; (iii) of the four possible configurations E, aE, Eb and aEb, only the double-occupied enzyme is active; (iv) the metal ion activation is a time-dependent process; (v) the dissociation constants for both the structural and catalytic sites may be identical or may differ by one or higher orders of magnitude; (vi) metal ion binding is stronger in the order Mn2+ greater than Co2+ much greater than Mg2+; (vii) pronounced increases in Km values concomitant with decreasing equivalents of metal ion added are only observed in the presence of Mg2+ ions.

Kinetic studies of Mg(2+)-, Co(2+)- and Mn(2+)-activated D-xylose isomerases.

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.

Localization of the essential histidine and carboxylate group in D-xylose isomerases.

D-Xylose isomerases from different bacterial strains were chemically modified with histidine and carboxylate-specific reagents. The active-site residues were identified by amino acid sequence analysis of peptides recognized by differential peptide mapping on ligand-protected and unprotected derivatized enzyme. Both types of modified residues were found to cluster in a region with consensus sequence: Phe-His-Asp-Xaa-Asp-Xaa-Xaa-Pro-Xaa-Gly, conserved in all D-xylose isomerases studied so far. These results are consistent with the recently published X-ray data of the enzyme active centre from Streptomyces rubiginosus showing hydrogen bond formation between Asp-57 and His-54 which locks the latter in one tautomeric form. A study of the pH-dependence of the kinetic parameters suggests the participation of a histidine group in the substrate-binding but not in the isomerization process. Comparison of the N-terminal amino acid sequences of several D-xylose isomerases further revealed a striking homology among the Actinomycetaceae enzymes and identifies them as a specific class of D-xylose isomerases.

Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule.

Reaction of Woodward's reagent K with D-xylose isomerases. Modification of an active site carboxylate residue.

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.

Evidence for an essential histidine residue in D-xylose isomerases.

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

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.

Catalytic versatility of Bacillus pumilus beta-xylosidase: glycosyl transfer and hydrolysis promoted with alpha- and beta-D-xylosyl fluoride.

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)

The pH dependence and group modification of beta-D-xylosidase from Bacillus pumilus: evidence for sulfhydryl and histidyl groups.

The pH dependence of the kinetic parameters of beta-D-xylosidase (EC. 3.2.1.37) from Bacillus pumilus reveals that an acidic functional group with pK 8.0 is involved in the catalysis. The fast inactivation of the dimeric enzyme by near equivalent amounts of methylmethanethiolsulfonate indicates that one thiol group per monomer is essential for catalysis, consistent with previously reported results. From the reactivity of the thiol groups with respect to 5,5'-dithiobis(2-nitrobenzoic acid), the absence of subunit cooperativity was indicated. The present study also reports on the inactivation of the enzyme by diethylpyrocarbonate, and provides evidence of the importance of a histidine residue. A mechanism of catalysis is presented, in which the thiol group interacts with the substrate via partial proton transfer. The mode of participation of the histidine group is difficult to specify, but may be associated with the maintenance of the active conformation of the enzyme.

Binding of alkyl beta-D-xylopyranosides, containing branched-chain, cyclic, and substituted aglycon groups, to beta-D-xylosidase from Bacillus pumilus PRL B12.

For a number of alkyl beta-D-xylopyranosides having branched-chain, cyclic, and substituted aglycon groups, and binding to beta-D-xylosidase from B. pumilus PRL B12, the binding constant Ki and (for some of them) the thermodynamic equilibrium parameters delta H0, delta S0, and delta G0 have been determined. Although the aglycon is bound through hydrophobic forces, no simple relationships between the binding parameters and the relative hydrophobicity of the alkyl beta-D-xylopyranosides could be demonstrated. All of the available evidence suggests that the aglycon sub-site has a highly specific structure which forces the atoms of the aglycon group to occupy well-defined positions. The supplementary energy-requirements resulting from the imposed restrictions seem to be the main reason for the irregular way in which the binding parameters depend on the aglycon structure.

Binding of n-alkyl beta-D-xylopyranosides and n-alkyl 1-thio-beta-D-xylopyranosides to beta-D-xylosidase from Bacillus pumilus PRL B12.

The binding of D-xylose and of a series of n-alkyl beta-D-xylopyranosides and their 1-thio analogues to beta-D-xylosidase from B. pumilus PRL B12 has been investigated. The binding constants and thermodynamic equilibrium parameters delta H0 and delta S0 have been determined. The enzyme does not distinguish between alpha- and beta-D-xylopyranose. Although the enthalpy of binding of D-xylose is very favourable, the overall free-energy is small, due to a large decrease in entropy. Furthermore, all of the evidence available suggests that the aglycon group is bound by unspecific, hydrophobic forces. However, simple correlations between the binding parameters and the relative hydrophobicity of the compounds could not be found. Unexpectedly, no parallelism between binding of n-alkyl beta-D-xylopyranosides and the corresponding 1-thio derivatives was found.

beta-D-xylosidase from Bacillus pumilus PRL B12: hydrolysis of aryl beta-D-xylopyranosides.

The influence of substituents on the binding and hydrolysis of several substituted beta-D-xylopyranosides by beta-D-xylosidase from Bacillus pumilus PRL B12 has been investigated. From a comparison of the inhibition constants of 1-thio-beta-D-xylopyranosides with the apparent Michaelis-Menten constants of the substrates, it followed that the latter constants are good approximations of the true equilibrium constants. The influence of the substituent on the rate and activation parameters is small. The results are in agreement with, but do not prove, a one-step mechanism without the formation of a glycosyl-enzyme intermediate.