Biomedical subjects
Publications and source records attributed to Stephen G Withers.
The atomic-resolution structure of a xylobiose-derived isofagomine lactam in complex with the xylanase Xyn10A from Streptomyces lividans reveals that the lactam is bound to the enzyme as the amide tautomer, with "reversed" protonation-states for nucleophile and acid-base.
The xylanase, BadX, from the alkalophilic Bacillus agaradhaerens was cloned, expressed and studied in comparison to a related family 11 xylanase, BcX, from B. circulans. Despite the alkaline versus neutral conditions under which these bacteria grow, BadX and BcX both exhibit optimal activity near pH 5.6 using the substrate o-nitrophenyl beta-xylobioside. Analysis of the bell-shaped activity profile of BadX yielded apparent pK(a) values of 4.2 and 7.1, assignable to its nucleophile Glu94 and general acid Glu184, respectively. In addition to having an approximately 10-fold higher k(cat)/K(m) value with this substrate at pH 6 and 40 degrees C, BadX has significantly higher thermal stability than BcX under neutral and alkaline conditions. This enhanced stability, rather than a shift in its pH-optimum, may allow BadX to hydrolyze xylan under conditions of elevated temperature and pH.
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The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).
Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.
Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism resulting in disaccharides or oligosaccharides with delta4,5-unsaturated uronic acid residues at their nonreducing end. Mechanistic details concerning the ordering of the bond-breaking and -forming steps of this enzymatic reaction are nonexistent, mainly due to the inhomogeneous nature of the polymeric substrates. The creation of a new class of synthetic substrates for this enzyme has allowed the measurement of defined and reproducible k(cat) and K(m) values and has expanded the range of mechanistic studies that can be performed. The primary deuterium kinetic isotope effect upon k(cat)/K(m) for the abstraction of the proton alpha to the carboxylic acid was measured to be 1.67 +/- 0.07, showing that deprotonation occurs in a rate-limiting step. Using substrates with leaving groups of differing reactivity, a flat linear free energy relationship was produced, indicating that the C4-O4 bond is not broken in a rate-determining step. Taken together, these results strongly suggest a stepwise mechanism. Consistent with this was the measurement of a secondary deuterium kinetic isotope effect upon k(cat)/K(m) of 1.01 +/- 0.03 on a 4-[(2)H]-substrate, indicating that no sp2 character is developed at C4 during the rate-limiting step, thereby ruling out a concerted syn-elimination.
SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site of SpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-acetamido group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-acetamido group preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-acetamido group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.
The catalytic mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) from family 39 of glycoside hydrolases has been subjected to a detailed kinetic investigation using a range of substrates. The enzyme exhibits a bell-shaped pH dependence of k(cat)/K(m), reflecting apparent pK(a) values of 4.1 and 6.8. The k(cat) and k(cat)/K(m) values for a series of aryl xylosides have been measured and used to construct two Brønsted plots. The plot of log(k(cat)/K(m)) against the pK(a) of the leaving group reveals a significant correlation (beta(lg) = -0.97, r(2) = 0.94, n = 8), indicating that fission of the glycosidic bond is significantly advanced in the transition state leading to the formation of the xylosyl-enzyme intermediate. The large negative value of the slope indicates that there is relatively little proton donation to the glycosidic oxygen in the transition state. A biphasic, concave-downward plot of log(k(cat)) against pK(a) provides good evidence for a two-step double-displacement mechanism involving a glycosyl-enzyme intermediate. For activated leaving groups (pK(a) < 9), the breakdown of the xylosyl-enzyme intermediate is the rate-determining step, as indicated by the absence of any effect of the pK(a) of the leaving group on log(k(cat)) (beta(lg) approximately 0). However, a strong dependence of the first-order rate constant on the pK(a) value of relatively poor leaving groups (pK(a) > 9) suggests that the xylosylation step is rate-determining for these substrates. Support for the dexylosylation chemical step being rate-determining for activated substrates comes from nucleophilic competition experiments in which addition of dithiothreitol results in an increase in turnover rates. Normal secondary alpha-deuterium kinetic isotope effects ((alpha-D)(V) or (alpha-D)(V/K) = 1.08-1.10) for three different substrates of widely varying pK(a) value (5.15-9.95) have been measured and these reveal that the transition states leading to the formation and breakdown of the intermediate are similar and both steps involve rehybridization of C1 from sp(3) to sp(2). These results are consistent only with "exploded" transition states, in which the saccharide moiety bears considerable positive charge, and the intermediate is a covalent acylal-ester where C1 is sp(3) hybridized.
The catalytic mechanism of the family 39 Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) involves a two-step double-displacement mechanism in which a covalent alpha-xylosyl-enzyme intermediate is formed with assistance from a general acid and then hydrolyzed with assistance from a general base. Incubation of recombinant XynB with the newly synthesized active site-directed inhibitor, N-bromoacetyl-beta-D-xylopyranosylamine, resulted in rapid, time-dependent inactivation of the enzyme (k(i)/K(i) = 4.3 x 10(-4) s(-1)mM(- 1)). Protection from inactivation using xylose or benzyl 1-thio-beta-xyloside suggested that the inactivation was active site-directed. Mass spectrometric analysis indicated that incubation of the enzyme with the inactivator resulted in the stoichiometric formation of a new enzyme species bearing the label. Comparative mapping of peptic digests of both the labeled and unlabeled enzyme by HPLC coupled to an electrospray ionization mass spectrometer permitted the identification of a labeled peptide. Sequencing of this peptide by tandem mass spectrometry identified Glu160 within the sequence (157)IWNEPNL(164) as the site of attachment of the N-acetyl-beta-D-xylopyranosylamine moiety. Kinetic analysis of the Glu160Ala mutant strongly suggests that this residue is involved in acid/base catalysis as follows. First, a significant difference in the dependence of k(cat)/K(m) on pH as compared to that seen for the wild-type enzyme was found, as expected for a residue that is involved in acid/base catalysis. The changes, however, were not as simple as those seen in other cases. Second, a dramatic decrease (up to 10(5)-fold) in the catalytic efficiency (k(cat)/K(m)) of the enzyme with a substrate requiring protonic assistance is observed upon such mutation. In contrast, the catalytic efficiency of the enzyme with substrates bearing a good leaving group, not requiring acid catalysis, is only moderately impaired relative to that of the wild-type enzyme (8-fold). Surprisingly, however, the glycosylation step was rate-limiting for all but the most reactive substrates. Last, the addition of azide as a competitive nucleophile resulted in the formation of a beta-xylosyl azide product and increased the k(cat) and K(m) values up to 8-fold while k(cat)/K(m) remained relatively unchanged. Such kinetic behavior is consistent with azide acting competitively with water as a nucleophile in the second step of the enzyme catalyzed reaction involving breakdown of the xylosyl-enzyme intermediate. Together, these results provide strong evidence for a role of Glu160 in acid/base catalysis but suggest that it may be partnered by a second carboxylic acid residue and that the enzyme may function through using acid catalysis involving reverse protonation of active site residues.
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Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism, resulting in disaccharides or oligosaccharides with Delta4,5-unsaturated uronic acid residues at their nonreducing end. The syntheses and testing of two potential inhibitors of this lyase are described. Methyl O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1-->4)-alpha-L-threo-hex-4-enopyranoside, 1, has the trigonal geometry at C5 of the uronic acid moiety expected at the transition state, yet retains the "leaving group" sugar moiety. Surprisingly, compound 1 showed no inhibition of the enzyme. The novel 5-nitro sugar, phenyl (5S)-5-nitro-beta-D-xylopyranoside, 2, is a monosaccharide nitro analogue of the natural substrate, with C5 being a carbon acid of pK(a) 8.8. The rate of reprotonation of the anion generated at this center is sufficiently low that the anion of 2 can be used directly in initial steady-state velocity measurements without significant interference from the conjugate carbon acid. The anion of compound 2 was found to be a competitive inhibitor with a K(i) value of 5 mM, whereas the conjugate acid had a K(i) value of 35 mM.
Enzymatic approaches for the preparation of oligosaccharides are interesting alternatives to traditional chemical synthesis, the main advantage being the regio- and stereoselectivity offered without the need for protecting groups. The use of solid-phase techniques offers easy workup procedures and the prospect of automatability. Here, we report the first application of glycosynthases to solid-phase oligosaccharide synthesis by use of the 51 kDa serine and glycine mutants of Agrobacterium sp. beta-glucosidase, Abg E358S and E358G. Acceptors were linked to PEGA resin through a backbone amide linker (BAL), and using these mutated enzymes, a galactose moiety was transferred from a donor sugar, alpha-D-galactosyl fluoride, with high efficiency (>90%) together with excellent recovery of material. Furthermore, it was demonstrated that a resin-bound model glycopeptide was also an acceptor for the glycosynthase.
alpha-Glucan lyase (EC 4.2.2.13, GLase) cleaves alpha-1,4-glucosidic bonds via an elimination reaction to produce 1,5-d-anhydrofructose. GLase was inactivated by the mechanism based alpha-glucosidase inactivator, 5-fluoro-beta-l-idosyl fluoride. The trapped glycosyl-enzyme intermediate was isolated and the nucleophilic amino acid residue (Asp 553) identified is equivalent to the residue so identified in sequence-related alpha-glucosidases. This intermediate undergoes a syn-elimination reaction to release the product. Further evidence against a direct trans-elimination mechanism was provided by the absence of a primary kinetic isotope effect on the substrate substituted with deuterium at the C2 position.
Lipopolysaccharyl alpha-galactosyltransferase from Neisseria meningitidis catalyzes the transfer of a galactosyl moiety from the activated donor UDP-Gal to glycoconjugates to yield an elongated saccharide product with net retention of anomeric configuration relative to the donor substrate. Through kinetic analyses in which the concentrations of both substrates are independently varied and through inhibition studies with dead-end analogues of both substrates and with the oligosaccharide product, we have demonstrated that this enzyme follows an ordered bi-bi kinetic mechanism. Various aspects of the chemical mechanism including the possible formation of a covalent glycosyl-enzyme intermediate were also probed using an assortment of strategies. While the results of these investigations were unable to clearly delineate the chemical mechanism of this enzyme, they provide important insights into the catalytic machinery surrounding the events involved in catalysis.
The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.
The compound (methylenecyclopropyl)formyl-CoA (MCPF-CoA) has been reported earlier as a potent active site-directed inactivator of bovine liver enoyl-CoA hydratase (ECH). It is believed that the mechanism of inactivation involves the attack of Cys114 at C-2' of MCPF-CoA, resulting in ring cleavage and permanent covalent modification of the enzyme. Here, we describe studies with the C114A mutant of bovine liver ECH, which was constructed and purified to determine the role of this residue in the catalytic mechanism of the enzyme. The C114A mutant, which is catalytically competent, shows an unexpected susceptibility to inactivation by MCPF-CoA, indicating that Cys114 is not the primary nucleophile responsible for the inactivation of the enzyme. To determine if catalytic residues Glu115 and Glu135 play a role in the inactivation of the enzyme, the E115Q and E135Q mutants were also constructed and purified. It was determined that these mutants did not react with MCPF-CoA, indicating a possible role for both residues in the inactivation of the wild-type enzyme. Pepsin digestion and subsequent LC-MS/MS analysis of the inactivated wild-type enzyme and C114A mutant revealed that Glu115 was modified in each case, supporting the hypothesis that this residue is the true nucleophile that traps MCPF-CoA and indicating that the covalent modification of Cys114 reported earlier may be a postinactivation artifact. We propose a modified mechanism of inactivation involving Glu115 and Glu135, and suggest that MCPF-CoA may be a mechanism-based inhibitor for bovine liver ECH.
Human pancreatic alpha-amylase (HPA) is a member of the alpha-amylase family involved in the degradation of starch. Some members of this family, including HPA, require chloride for maximal activity. To determine the mechanism of chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q) were made in which residues in the chloride ion binding site were replaced. Mutations in this binding site were found to severely affect the ability of HPA to bind chloride ions with no binding detected for the R195 and R337 mutant enzymes. X-ray crystallographic analysis revealed that these mutations did not result in significant structural changes. However, the introduction of these mutations did alter the kinetic properties of the enzyme. Mutations to residue R195 resulted in a 20-450-fold decrease in the activity of the enzyme toward starch and shifted the pH optimum to a more basic pH. Interestingly, replacement of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer required chloride for catalysis and has a pH profile similar to that of wild-type HPA. In contrast, a mutation at residue N298 resulted in an enzyme that had much lower binding affinity for chloride but still required chloride for maximal activity. We propose that the chloride is required to increase the pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the presence of R337, a positively charged residue.
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