Mapping the conformational itinerary of beta-glycosidases by X-ray crystallography.

The conformational agenda harnessed by different glycosidases along the reaction pathway has been mapped by X-ray crystallography. The transition state(s) formed during the enzymic hydrolysis of glycosides features strong oxocarbenium-ion-like character involving delocalization across the C-1-O-5 bond. This demands planarity of C-5, O-5, C-1 and C-2 at or near the transition state. It is widely, but incorrectly, assumed that the transition state must be (4)H(3) (half-chair). The transition-state geometry is equally well supported, for pyranosides, by both the (4)H(3) and (3)H(4) half-chair and (2,5)B and B(2,5) boat conformations. A number of retaining beta-glycosidases acting on gluco -configured substrates have been trapped in Michaelis and covalent intermediate complexes in (1)S(3) (skew-boat) and (4)C(1) (chair) conformations, respectively, pointing to a (4)H(3)-conformed transition state. Such a (4)H(3) conformation is consistent with the tight binding of (4)E- (envelope) and (4)H(3)-conformed transition-state mimics to these enzymes and with the solution structures of compounds bearing an sp (2) hybridized anomeric centre. Recent work reveals a (1)S(5) Michaelis complex for beta-mannanases which, together with the (0)S(2) covalent intermediate, strongly implicates a B(2,5) transition state for beta-mannanases, again consistent with the solution structures of manno -configured compounds bearing an sp (2) anomeric centre. Other enzymes may use different strategies. Xylanases in family GH-11 reveal a covalent intermediate structure in a (2,5)B conformation which would also suggest a similarly shaped transition state, while (2)S(0)-conformed substrate mimics spanning the active centre of inverting cellulases from family GH-6 may also be indicative of a (2,5)B transition-state conformation. Work in other laboratories on both retaining and inverting alpha-mannosidases also suggests non-(4)H(3) transition states for these medically important enzymes. Three-dimensional structures of enzyme complexes should now be able to drive the design of transition-state mimics that are specific for given enzymes, as opposed to being generic or merely fortuitous.

Dissection of nucleophilic and acid-base catalysis in glycosidases.

A startling array of added anions have been observed to function as replacement catalytic nucleophiles in mutant glycosidases, including formate, azide, fluoride and other halides. Likewise, the mechanism of acid-base catalysis is somewhat plastic. The carboxylic acids can be substituted by a sulfenic acid or by ascorbate, and the effective acid strength enhanced by the introduction of strong hydrogen bonds. These studies provide an interesting bridge between enzymes and models thereof.

Glycosidase mechanisms: anatomy of a finely tuned catalyst.

In order to accelerate the hydrolysis of glycosidic bonds by factors approaching 10(17)-fold, glycosidases have evolved finely tuned active sites optimally configured for transition-state stabilization. Structural analyses of various enzyme complexes representing stable intermediates along the reaction coordinate, in conjunction with detailed mechanistic studies on wild-type and mutant enzymes, have delineated the contributions of nucleophilic and general acid/base catalysis, as well as the roles of noncovalent interactions, to these impressive rate enhancements.

The E358S mutant of Agrobacterium sp. beta-glucosidase is a greatly improved glycosynthase.

Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The 'original' glycosynthase, derived from Agrobacterium sp. beta-glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q., Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583-5584]. This mutant has now been re-cloned with a His(6)-tag into a pET-29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24-fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor acceptors for Abg E358A, such as PNP-GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP-beta-LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the alpha-glycosyl fluoride.

Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase celB.

Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cellulose with retention of anomeric configuration. A recent X-ray structure of the catalytic domain at 1.75 A resolution has led to the preliminary assignment of Glu-120 and Glu-203 as the catalytic nucleophile and general acid-base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davies (1997) Biochemistry 36, 16032-16039]. The present study confirms the identity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturable fashion, yielding the parameters kinact=0.29+/-0.02 min-1 and Kinact=0.72+/-0.08 mM. Uncompetitive inhibition was observed at high concentrations of 2FDNPC (Ki=9+/-1 mM), a behaviour that was also observed with the substrate 2',4'-dinitrophenyl beta-D-cellobioside (kcat=40+/-1 s-1, Km=0.35+/-0.03 mM, Ki=24+/-4 mM). Protection against inactivation was afforded by the competitive inhibitor cellobiose. The electrospray ionization (ESI) mass spectrum of the intact labelled CelB indicated that the inactivator had labelled the enzyme stoichiometrically. Reactivation of the trapped intermediate occurred spontaneously (kH2O=0.0022 min-1) or via transglycosylation, with cellobiose acting as an acceptor ligand (kreact=0.024 min-1, Kreact=54 mM). Digestion of the labelled enzyme by pepsin followed by LC-ESI-tandem MS (MS-MS) operating in neutral loss mode identified a labelled, singly charged peptide of m/z 947.5 Da. Isolation of this peptide by HPLC and subsequent collision-induced fragmentation by ESI-MS-MS produced a daughter-ion spectrum that corresponded to a sequence (QTEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid-base catalyst Glu-203 are conserved in all known family-12 sequences.

Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry.

For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-d-xylobioside was detected by time-resolved ESI-MS. The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic data are in agreement with those determined by monitoring the release of 2, 5-dinitrophenol with stopped-flow UV-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.