Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.

The number of all possible linear and branched isomers of a hexasaccharide was calculated and found to be > 1.05 x 10(12). This large number defines the Isomer Barrier, a persistent technological barrier to the development of a single analytical method for the absolute characterization of carbohydrates, regardless of sample quantity. Because of this isomer barrier, no single method can be employed to determine complete oligosaccharide structure in 100 nmol amounts with the same assurance that can be achieved for 100 pmol amounts with single-procedure Edman peptide or Sanger DNA sequencing methods. Difficulties in the development of facile synthetic schemes for oligosaccharides are also explained by this large number. No current method of chemical or physical analysis has the resolution necessary to distinguish among 10(12) structures having the same mass. Therefore the 'characterization' of a middle-weight oligosaccharide solely by NMR or mass spectrometry necessarily contains a very large margin of error. Greater uncertainty accompanies results performed solely by sequential enzyme degradation followed by gel-permeation chromatography or electrophoresis, as touted by some commercial advertisements. Much of the literature which uses these single methods to 'characterize' complex carbohydrates is, therefore, in question, and journals should beware of publishing structural characterizations unless the authors reveal all alternate possible structures which could result from their analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

A caste structure is maintained in termite societies and juvenile hormone (JH) is generally regarded as the most important regulator in these termite colonies. Here, we demonstrate that the soldier caste regulates JH in workers of Coptotermes formosanus Shiraki. Worker termites (80 Ð100 individuals) were placed in petri dishes with 0, 5, 10, or 20% soldiers. JH III titers of groups of these workers were monitored at 14, 28, 42, and 56 d. Any changes in soldier caste proportions also were noted at each sample date. On the Þrst sample date, the JH levels in workers were similar among treatments with different initial soldier proportions, and no new soldiers were formed. Over the next three sample dates, the worker JH levels were higher for low initial soldier proportion treatments and vice versa. Concurrently, soldier formation increased with lower initial soldier proportions. JH titers in workers showed a positive and statistically signiÞcant relationship to soldier numbers until a certain soldier proportion was reached. These results provide evidence that soldier caste proportions regulate JH levels and thereby caste differentiation in workers. The means by which this regulatory mechanism may proceed is discussed.

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