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Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucosebinding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the ¹H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the ¹H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Enzymes are called upon to differ greatly in the difficulty of the tasks that they perform. The catalytic proficiency of an enzyme can be evaluated by comparing the second-order rate constant ( k cat / K m ) with the rate of the spontaneous reaction in neutral solution in the absence of a catalyst. The proficiencies of enzymes, measured in this way, are matched by their affinity constants for the altered substrate in the transition state. These values vary from approximately ∼10 9 M −1 for carbonic anhydrase to ∼10 23 M −1 for yeast orotidine 5′-phosphate decarboxylase (ODCase). ODCase turns its substrate over with a half-time of 18 ms, in a reaction that proceeds in its absence with a half-time of 78 million years in neutral solution. ODCase differs from other decarboxylases in that its catalytic activity does not depend on the presence of metals or other cofactors, or on the formation of a covalent bond to the substrate. Several mechanisms of transition state stabilization are considered in terms of ODCase crystal structures observed in the presence and absence of bound analogs of the substrate, transition state, and product. Very large connectivity effects are indicated by the results of experiments testing how transition state stabilization is affected by the truncation of binding determinants of the substrate and the active site.

The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic β-cells. Photochemical crosslinking studies using a synthetic analog of BAD's BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the K0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.

Objective: To compare levels of particulate matter, as a marker of secondhand smoke (SHS) levels, in pubs before and 2 months after the implementation of Scottish legislation to prohibit smoking in substantially enclosed public places. Design: Comparison of SHS levels before and after the legislation in a random selection of 41 pubs in 2 Scottish cities. Methods: Fine particulate matter <2.5 μm in diameter (PM2.5) was measured discreetly for 30 min in each bar on 1 or 2 visits in the 8 weeks preceding the starting date of the Smoking, Health and Social Care (Scotland) Act 2005 and then again 2 months after the ban. Repeat visits were undertaken on the same day of the week and at approximately the same time of the day. Results: PM2.5 levels before the introduction of the legislation averaged 246 μg/m3 (range 8–902 μg/m3). The average level reduced to 20 μg/m3 (range 6–104 μg/m3) in the period after the ban. Levels of SHS were reduced in all 53 post-ban visits, with the average reduction being 86% (range 12–99%). PM2.5 concentrations in most pubs post-ban were comparable to the outside ambient air PM2.5 level. Conclusions: This study has produced the largest dataset of pre- and post-ban SHS levels in pubs of all worldwide smoke-free legislations introduced to date. Our results show that compliance with the Smoking, Health and Social Care (Scotland) Act 2005 has been high and this has led to a marked reduction in SHS concentrations in Scottish pubs, thereby reducing both the occupational exposure of workers in the hospitality sector and that of non-smoking patrons.

Microbial niches contain toxic chemicals capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival in the presence of a normally lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. We also screened a comprehensive library of E. coli single-gene deletion mutants and identified 63 strains displaying increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene encoding a predicted glutathione transferase. Herein, YliJ is shown to catalyze the glutathione-dependent dehalogenation of bromoacetate with a k cat /K m value of 5.4 × 10³ M⁻¹ s⁻¹. YliJ displays exceptional substrate specificity and produces a rate enhancement exceeding 5 orders of magnitude, remarkable characteristics for reactivity with a nonnatural molecule. This study illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins.

The ROK (repressor, open reading frame, kinase) protein family (Pfam 00480) is a large collection of bacterial polypeptides that includes sugar kinases, carbohydrate responsive transcriptional repressors, and many functionally uncharacterized gene products. ROK family sugar kinases phosphorylate a range of structurally distinct hexoses including the key carbon source d-glucose, various glucose epimers, and several acetylated hexosamines. The primary sequence elements responsible for carbohydrate recognition within different functional categories of ROK polypeptides are largely unknown due to a limited structural characterization of this protein family. In order to identify the structural bases for substrate discrimination in individual ROK proteins, and to better understand the evolutionary processes that led to the divergent evolution of function in this family, we constructed an inclusive alignment of 227 representative ROK polypeptides. Phylogenetic analyses and ancestral sequence reconstructions of the resulting tree reveal a discrete collection of active site residues that dictate substrate specificity. The results also suggest a series of mutational events within the carbohydrate-binding sites of ROK proteins that facilitated the expansion of substrate specificity within this family. This study provides new insight into the evolutionary relationship of ROK glucokinases and non-ROK glucokinases (Pfam 02685), revealing the primary sequence elements shared between these two protein families, which diverged from a common ancestor in ancient times.

Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity-onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder-order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder-order cycle of the small domain as a \"time-delay loop,\" which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.

Abstract A key step in metabolic pathway evolution is the recruitment of promiscuous enzymes to perform new functions. Despite the recognition that promiscuity is widespread in biology, factors dictating the preferential recruitment of one promiscuous enzyme over other candidates are unknown. Escherichia coli contains four sugar kinases that are candidates for recruitment when the native glucokinase machinery is deleted—allokinase (AlsK), manno(fructo)kinase (Mak), N-acetylmannosamine kinase (NanK), and N-acetylglucosamine kinase (NagK). The catalytic efficiencies of these enzymes are 103- to 105-fold lower than native glucokinases, ranging from 2,400 M−1 s−1 for the most active candidate, NagK, to 15 M−1 s−1 for the least active candidate, AlsK. To investigate the relationship between catalytic activities of promiscuous enzymes and their recruitment, we performed adaptive evolution of a glucokinase-deficient E. coli strain to restore glycolytic metabolism. We observed preferential recruitment of NanK via a trajectory involving early mutations that facilitate glucose uptake and amplify nanK transcription, followed by nonsynonymous substitutions in NanK that enhance the enzyme's promiscuous glucokinase activity. These substitutions reduced the native activity of NanK and reduced organismal fitness during growth on an N-acetylated carbon source, indicating that enzyme recruitment comes at a cost for growth on other substrates. Notably, the two most active candidates, NagK and Mak, were not recruited, suggesting that catalytic activity alone does not dictate evolutionary outcomes. The results highlight our lack of knowledge regarding biological drivers of enzyme recruitment and emphasize the need for a systems-wide approach to identify factors facilitating or constraining this important adaptive process.

ObjectivesIn the 1950s the Pneumoconiosis Field Research (PFR) programme was set up to study the health of British coal workers. Studies included regular health surveys, an intensive characterisation of workers' individual exposures, and entry to a cohort followed up to the present for cause-specific mortality. This study reports on analyses of cause-specific mortality in a cohort of almost 18 000 men from 10 British collieries.MethodsExternal analyses used standardised mortality ratios (SMRs), comparing observed mortality with reference rates from the regions in which the collieries were situated. Causes investigated include lung and stomach cancers, chronic obstructive pulmonary disease and cardiovascular endpoints. Internal analyses used Cox regression models with time-dependent exposures adjusting for the confounding effects of age, smoking, cohort entry date and regional differences in population mortality rates.ResultsSeveral causes showed evidence of a healthy worker effect early in the follow-up, with a deficit in the SMR diminishing over time. For most of the causes there was a significant excess in the latter part of follow-up.Internal analyses found evidence of an association between increased risks of lung cancer and increased quartz exposure, particularly at a lag of 15 years.Risks of mortality from non-malignant respiratory disease showed increases with increased exposure to respirable dust.ConclusionsThis paper adds to the evidence on the long-term effects of exposure to coalmine dust on mortality from respiratory diseases.

The tatanans are members of a novel class of complex sesquilignan natural products recently isolated from the rhizomes of Acorus tatarinowii Schott plants. Tatanans A, B and C have previously been reported to have potent glucokinase-activating properties that exceed the in vitro activity of known synthetic antidiabetic agents. Here, using a series of sequential [3,3]-sigmatropic rearrangements, we report the total synthesis of tatanan A in 13 steps and 13% overall yield. We also complete a concise enantioselective total synthesis of more complex, atropisomeric tatanans B and C via a distinct convergent strategy based on a palladium-catalysed diastereotopic aromatic group differentiation (12 steps, 4% and 8% overall yield, respectively). A plausible biosynthetic relationship between acyclic tatanan A and spirocyclic tatanans B and C is proposed and probed experimentally. With sufficient quantities of the natural products in hand, we undertake a detailed functional characterization of the biological activities of tatanans A–C. Contrary to previous reports, our assays utilizing pure recombinant human enzyme demonstrate that tatanans do not function as allosteric activators of glucokinase. Tatanans A, B and C, members of a family of complex sesquilignan natural products, were recently reported to possess potent anti-diabetic, glucokinase-activating properties. Here, a convergent enantioselective total synthesis of these tatanans enabled by catalytic allylic dearomatization is described. Contrary to previous reports, biological assays show that tatanans A–C are not allosteric activators of glucokinase.