Mechanism of GlvA from <i>Bacillus subtilis</i>: A Detailed Kinetic Analysis of a 6-Phospho-α-glucosidase from Glycoside Hydrolase Family 4
Bibliographic record
Abstract
GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis assigned to glycoside hydrolase family 4, catalyzes the hydrolysis of maltose 6'-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In contrast to previous reports and consistent with the proposed mechanism, GlvA is only activated in the presence of the nicotinamide cofactor in its oxidized, and not the reduced NADH, form. Significantly, GlvA catalyzes the hydrolysis of both 6-phospho-alpha- and 6-phospho-beta-glucosides containing activated leaving groups such as p-nitrophenol and does so with retention and inversion, respectively, of anomeric configuration. Mechanistic details of the individual bond cleaving and forming steps were probed using a series of 6-phospho-alpha- and 6-phospho-beta-glucosides. Primary deuterium kinetic isotope effects (KIEs) were measured for both classes of substrates in which either the C2 or the C3 protons have been substituted with a deuterium, consistent with C-H bond cleavage at each center being partially rate-limiting. Kinetic parameters were also determined for 1-[2H]-substituted substrates, and depending on the substrates and the reaction conditions, the measurements of kcat and kcat/KM produced either no KIEs or inverse KIEs. In conjunction with results of Brønsted analyses with both aryl 6-phospho-alpha- and beta-glucosides, the kinetic data suggest that GlvA utilizes an E1cb mechanism analogous to that proposed for the Thermotoga maritima BglT, a 6-phospho-beta-glucosidase in glycoside hydrolase family 4 (Yip, V.L.Y et al. (2006) Biochemistry 45, 571-580). The pattern of isotope effects measured and the observation of very similar kcat values for all substrates, including unactivated and natural substrates, indicate that the oxidation and deprotonation steps are rate-limiting steps in essentially all cases. This mechanism permits the cleavage of both alpha- and beta-glycosides within the same active site motif and, for activated substrates that do not require acid catalysis for cleavage, within the same active site, yielding the product sugar-6-phosphate in the same anomeric form in the two cases.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".