A simple kinetic test to distinguish exo-glucosidase and beta-glucosidase activities

Unusual kinetic behaviour was observed in assaying spectrophotometrically for exo-glucanase activity in a beta-glucosidase isolated from A. faecalis using p-nitrophenyl beta-cellobioside as substrate. At high substrate concentrations no phenol was released whereas at low concentrations a rapid release of phenol was detected and this increased in rate with extent of hydrolysis. These results are consistent with a model involving tight binding of the substrate to the enzyme and an initial exo-glucosidase-catalysed hydrolysis to produce glucose and p-nitrophenyl glucoside. Subsequent hydrolysis of the nitrophenyl glucoside results in phenol release, but only after sufficient concentrations have accumulated to compete with the cellobioside. This theory was confirmed by product analysis and by measuring the affinity of the substrate for the enzyme by its inhibition of p-nitrophenyl glucoside hydrolysis. Observation of such kinetic behaviour allows distinction between beta-glucosidase and exo-glucosidase activities.     

NMR spectroscopic characterization of a beta-(1,4)-glycosidase along its reaction pathway: stabilization upon formation of the glycosyl-enzyme intermediate

NMR spectroscopy was used to search for mechanistically significant differences between the thermodynamic and dynamic properties of the 34 kDa (alpha/beta)8-barrel catalytic domain of beta-(1,4)-glycosidase Cex (or CfXyn10A) in its free (apo-CexCD) and trapped glycosyl-enzyme intermediate (2FCb-CexCD) states. The main chain chemical shift perturbations due to the covalent modification of CexCD with the mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside are limited to residues within its active site. Thus, consistent with previous crystallographic studies, formation of the glycosyl-enzyme intermediate leads to only localized structural changes. Furthermore, 15N relaxation methods demonstrated that the backbone amide and tryptophan side chains of apo-CexCD are very well ordered on both the nanosecond to picosecond and millisecond to microsecond time scales and that these dynamic features also do not change significantly upon formation of the trapped intermediate. However, covalent modification of CexCD led to the increased protection of many amides and indoles, clustered around the active site of the enzyme, against fluctuations leading to hydrogen exchange. Similarly, thermal denaturation studies demonstrated that 2FCb-CexCD has a significantly higher midpoint unfolding temperature than apo-CexCD. The covalently modified protein also exhibited markedly increased resistance to proteolytic degradation by thermolysin relative to apo-CexCD. Thus, the local and global stability of CexCD increase along its reaction pathway upon formation of the glycosyl-enzyme intermediate, while its structure and fast time scale dynamics remain relatively unperturbed. This may reflect thermodynamically favorable interactions with the relatively rigid active site of Cex necessary to bind, distort, and subsequently hydrolyze glycoside substrates.     

Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.     

In vivo downregulation of protein synthesis in the snail Helix apersa during estivation

Protein synthesis is downregulated during metabolic depression in a number of systems where the metabolic depression is effected by obvious extrinsic cues. The metabolic depression of the estivating land snail Helix apersa occurs in the absence of any obvious physiological stress and has an intrinsic component independent of temperature, pH, O(2) status, or osmolality. We show that this metabolic depression is accompanied by a downregulation of protein synthesis in vivo. The rate of protein synthesis decreases in two major tissues during estivation: to 23% and 53% of the awake rate in hepatopancreas and foot muscle, respectively. We show from calculations of the theoretical contribution of protein synthesis to total O(2) consumption that the depression of protein synthesis must be a significant, obligate, in vivo component of metabolic depression in H. aspersa.     

Structural and kinetic analysis of two covalent sialosyl-enzyme intermediates on Trypanosoma rangeli sialidase

Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that catalyzes the cleavage of alpha-2-->3-linked sialic acid residues from sialoglycoconjugates with overall retention of anomeric configuration. Retaining glycosidases usually operate through a ping-pong mechanism, wherein a covalent intermediate is formed between the carbohydrate and an active site carboxylic acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the catalytic nucleophile, leaving the possibility of an essentially different catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a typical sialidase. Here we present the three-dimensional structures of the covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two different mechanism-based inactivators at 1.9 and 1.7 Angstroms resolution. To our knowledge, these are the first reported structures of enzymatically competent covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses have been carried out on the formation and turnover of both intermediates, showing that structural modifications to these inactivators can be used to modify the lifetimes of covalent intermediates. These results provide further evidence that all sialidases likely operate through a similar mechanism involving the transient formation of a covalently sialylated enzyme. Furthermore, we believe that the ability to "tune" the inactivation and reactivation rates of mechanism-based inactivators toward specific enzymes represents an important step toward developing this class of inactivators into therapeutically useful compounds.     

The cellulose-binding domains from Cellulomonas fimi beta-1, 4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations

The N-terminal cellulose-binding domains CBDN1 and CBDN2 from Cellulomonas fimi cellulase CenC each adopt a jelly-roll beta-sandwich structure with a cleft into which amorphous cellulose and soluble cellooligosaccharides bind. To determine the orientation of the sugar chain within these binding clefts, the association of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl) spin-labeled derivatives of cellotriose and cellotetraose with isolated CBDN1 and CBDN2 was studied using heteronuclear 1H-15N NMR spectroscopy. Quantitative binding measurements indicate that the TEMPO moiety does not significantly perturb the affinity of the cellooligo-saccharide derivatives for the CBDs. The paramagnetic enhancements of the amide 1HN longitudinal (DeltaR1) and transverse (DeltaR2) relaxation rates were measured by comparing the effects of TEMPO-cellotetraose in its nitroxide (oxidized) and hydroxylamine (reduced) forms on the two CBDs. The bound spin-label affects most significantly the relaxation rates of amides located at both ends of the sugar-binding cleft of each CBD. Similar results are observed with TEMPO-cellotriose bound to CBDN1. This demonstrates that the TEMPO-labeled cellooligosaccharides, and by inference strands of amorphous cellulose, can associate with CBDN1 and CBDN2 in either orientation across their beta-sheet binding clefts. The ratio of the association constants for binding in each of these two orientations is estimated to be within a factor of five to tenfold. This finding is consistent with the approximate symmetry of the hydrogen-bonding groups on both the cellooligosaccharides and the residues forming the binding clefts of the CenC CBDs.     

Megabase Length Hypermutation Accompanies Human Structural Variation at 17p11.2

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Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions

Background  

The pan-genome of a bacterial species consists of a core and an accessory gene pool. The accessory genome is thought to be an important source of genetic variability in bacterial populations and is gained through lateral gene transfer, allowing subpopulations of bacteria to better adapt to specific niches. Low-cost and high-throughput sequencing platforms have created an exponential increase in genome sequence data and an opportunity to study the pan-genomes of many bacterial species. In this study, we describe a new online pan-genome sequence analysis program, Panseq.     

Effect of exercise intensity and duration on postexercise metabolism

Data are reported on the net recovery O2 consumption (VO2) for nine male subjects (mean age 21.9 yr, VO2max 63.0 ml.kg-1.min-1, body fat 10.6%) used in a 3 (independent variables: intensities of 30, 50, and 70% VO2max) x 3 (independent variables: durations of 20, 50, and 80 min) repeated measures design (P less than or equal to 0.05). The 8-h mean excess postexercise O2 consumptions (EPOCs) for the 20-, 50-, and 80-min bouts, respectively, were 1.01, 1.43, and 1.04 liters at 30% VO2max (6.8 km/h); 3.14, 5.19, and 6.10 liters at 50% VO2max (9.5 km/h); and 5.68, 10.04, and 14.59 liters at 70% VO2max (13.4 km/h). The mean net total O2 costs (NTOC = net exercise VO2 + EPOC) for the 20-, 50-, and 80-min bouts, respectively, were 20.48, 53.20, and 84.23 liters at 30% VO2max; 38.95, 100.46, and 160.59 liters at 50% VO2max; and 58.30, 147.48, and 237.17 liters at 70% VO2max. The nine EPOCs ranged only from 1.0 to 8.9% of the NTOC (mean 4.8%) of the exercise. These data, therefore, indicate that in well-trained subjects the 8-h EPOC per se comprises a very small percentage of the NTOC of exercise.