[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Cooperativity is widespread in biology. It empowers a variety of regulatory mechanisms and impacts both the kinetic and thermodynamic properties of macromolecular systems. Traditionally, cooperativity is viewed as requiring the participation of multiple, spatially distinct binding sites that communicate via ligand-induced structural rearrangements; however, cooperativity requires neither multiple ligand binding events nor multimeric assemblies. An underappreciated manifestation of cooperativity has been observed in the non-Michaelis-Menten kinetic response of certain monomeric enzymes that possess only a single ligand-binding site. In this review, we present an overview of kinetic cooperativity in monomeric enzymes. We discuss the primary mechanisms postulated to give rise to monomeric cooperativity and highlight modern experimental methods that could offer new insights into the nature of this phenomenon. We conclude with an updated list of single subunit enzymes that are suspected of displaying cooperativity, and a discussion of the biological significance of this unique kinetic response.
[show abstract][hide abstract] ABSTRACT: Glucokinase catalyzes the ATP-dependent phosphorylation of glucose, a chemical transformation that represents the rate-limiting step of glycolytic metabolism in the liver and pancreas. Glucokinase is a central regulator of glucose homeostasis as evidenced by its association with two disease states, maturity onset diabetes of the young (MODY) and persistent hyperinsulinemia of infancy (PHHI). Mammalian glucokinase is subject to homotropic allosteric regulation by glucose-the steady-state velocity of glucose-6-phosphate production is not hyperbolic, but instead displays a sigmoidal response to increasing glucose concentrations. The positive cooperativity displayed by glucokinase is intriguing since the enzyme functions as a monomer under physiological conditions and contains only a single binding site for glucose. Despite the existence of several models of kinetic cooperativity in monomeric enzymes, a consensus has yet to be reached regarding the mechanism of allosteric regulation in glucokinase. Experimental evidence collected over the last 45 years by a number of investigators supports a link between cooperativity and slow conformational reorganizations of the glucokinase scaffold. In this review, we summarize advances in our understanding of glucokinase allosteric regulation resulting from recent X-ray crystallographic, pre-equilibrium kinetic and high-resolution nuclear magnetic resonance investigations. We conclude with a brief discussion of unanswered questions regarding the mechanistic basis of kinetic cooperativity in mammalian glucokinase.
Archives of Biochemistry and Biophysics 11/2011; 519(2):103-11. · 3.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human pancreatic glucokinase is a monomeric enzyme that displays kinetic cooperativity, a feature that facilitates enzyme-mediated regulation of blood glucose levels in the body. Two theoretical models have been proposed to describe the non-Michaelis-Menten behavior of human glucokinase. The mnemonic mechanism postulates the existence of one thermodynamically favored enzyme conformation in the absence of glucose, whereas the ligand-induced slow transition model (LIST) requires a preexisting equilibrium between two enzyme species that interconvert with a rate constant slower than turnover. To investigate whether either of these mechanisms is sufficient to describe glucokinase cooperativity, a transient-state kinetic analysis of glucose binding to the enzyme was undertaken. A complex, time-dependent change in enzyme intrinsic fluorescence was observed upon exposure to glucose, which is best described by an analytical solution comprised of the sum of four exponential terms. Transient-state glucose binding experiments conducted in the presence of increasing glycerol concentrations demonstrate that three of the observed rate constants decrease with increasing viscosity. Global fit analyses of experimental glucose binding curves are consistent with a kinetic model that is an extension of the LIST mechanism with a total of four glucose-bound binary complexes. The kinetic model presented herein suggests that glucokinase samples multiple conformations in the absence of ligand and that this conformational heterogeneity persists even after the enzyme associates with glucose.
[show abstract][hide abstract] ABSTRACT: 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 × 10(3) 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.
Proceedings of the National Academy of Sciences 10/2010; 107(42):17968-73. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of human glucokinase, a 52 kDa monomeric enzyme that displays kinetic cooperativity. (1)H-(15)N transverse relaxation optimized spectra of uniformly labeled glucokinase, recorded in the absence and presence of glucose, reveal significant cross-peak overlap and heterogeneous peak intensities that persist over a range of temperatures. (15)N-specific labeling of isoleucines and tryptophans, reporting on backbone and side chain dynamics, respectively, demonstrates that both unliganded and glucose-bound enzymes sample multiple conformations, although glucose stabilizes certain conformations. These results provide the first direct evidence of glucokinase conformational heterogeneity and hence shed light on the molecular basis of cooperativity.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Molecular Evolution 06/2010; 70(6):545-56. · 2.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recently, we reported that YghZ from Escherichia coli functions as an efficient L-glyceraldehyde 3-phosphate reductase (Gpr). Here we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a K(d) value of 5.8+/-0.2 microM in the absence of NADPH and a K(d) value of 11+/-1.3 microM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the L-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K+ channel beta-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K+ channels with hemoprotein-like, O2-sensing properties. To date the molecular origin of O2 sensing by voltage-gated K+ channels is unknown and the results presented herein suggest a role for heme in this process.
[show abstract][hide abstract] ABSTRACT: Human glucokinase is a monomeric enzyme that displays a sigmoidal steady-state kinetic response toward increasing glucose concentrations. The allosteric regulation produced by glucose is postulated to arise from the slow interconversion of multiple enzyme conformations during the course of catalysis. Crystallographic data suggest that structural rearrangements linked to glucokinase cooperativity involve a substrate-induced repositioning of an alpha-helix (alpha13) located at the C-terminus of the polypeptide. Here, we show that removal of helix alpha13 abolishes cooperativity and restores Michaelis-Menten kinetics, while reducing the k(cat) value of the wild-type enzyme by 160-fold. The impaired catalytic activity of the truncated enzyme is not rescued by the trans addition of a synthetic alpha13 peptide. Unexpectedly, the K(m glucose) value of a glucokinase variant lacking alpha13 is equivalent to the K(0.5 glucose) value of the full-length enzyme. Glucokinase steady-state kinetics is unaffected by the elongation of alpha13 via the addition of a C-terminal polyalanine tail. To explore the link between cooperativity and the primary sequence of alpha13, we randomized seven residues within the helix core. Genetic selection experiments in a glucokinase-deficient bacterium identified a variety of hyperactive alpha13 variants that display lower K(0.5 glucose) values, Hill coefficients near unity, and enhanced equilibrium binding affinities for glucose. The present results demonstrate that alpha13 plays an essential role in facilitating cooperativity. Our findings also establish a link between the primary amino acid sequence of helix alpha13 and the functional dynamics of the glucokinase scaffold that are required for allostery.
[show abstract][hide abstract] ABSTRACT: We describe the discovery of 11 new activating mutations in the human glk gene associated with the disease persistent hyperinsulinemic hypoglycemia of infancy (PHHI). Three of the newly identified substitutions colocalize to a region of the glucokinase polypeptide where a synthetic allosteric activator binds. Of these substitutions, I211F is the most active variant identified to date, with a k(cat)/K(0.5,glucose) value (6.6 x 10(4) M(-1) s(-1)) that is 12-fold higher than that of wild-type glucokinase. The stimulatory mutations described herein represent surreptitious genetic determinants of PHHI. They also identify novel features of the glucokinase scaffold that could be targeted during the development of diabetes therapeutics.
[show abstract][hide abstract] ABSTRACT: Triosephosphate isomerase (TIM) catalyzes the interconversion of d-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the Escherichia coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of l-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate.
[show abstract][hide abstract] ABSTRACT: In 1995, Radzicka and Wolfenden reported that the rate enhancement produced by orotidine 5'-phosphate decarboxylase (ODCase) approaches 10(17), making this enzyme the most effective pure protein catalyst known in Nature [A. Radzicka, R. Wolfenden, Science 267 (1995) 90-93]. Over the last 12 years, there have been many hypotheses put forward to explain that impressive effect. In this perspective, we provide a summary of the reaction pathways under consideration for ODCase, highlight the supporting and refuting data, and suggest experiments designed to further test each of the candidate pathways.
[show abstract][hide abstract] ABSTRACT: The d-allose and N-acetyl-d-mannosamine kinases of Escherichia coli K-12 are divergent members of the functionally diverse ROK (repressor, open reading frame, kinase) superfamily. Previous work in our laboratory has demonstrated that AlsK and NanK possess weak phosphoryl transfer activity toward the alternate substrate d-glucose. To gain insight into the evolutionary mechanisms that fuel the specialization of individual enzyme function, experimental laboratory evolution was conducted to improve the glucokinase activities of AlsK and NanK. Error-prone PCR was combined with in vivo functional selection in a glucokinase-deficient bacterium to identify four independent single nucleotide substitutions in the alsK and nanK genes that improve the glucokinase activity of each enzyme. The most advantageous substitutions, L84P in NanK and A73G in AlsK, enhance the kcat/Km values for phosphoryl transfer to glucose by 12-fold and 60-fold, respectively. Both substitutions co-localize to a variable loop region located between the fourth beta-sheet and the second alpha-helix of the ROK scaffold. A multiple sequence alignment of diverse ROK family members reveals that the A73G substitution in AlsK recapitulates a conserved glycine residue present in many ROK proteins, including some transcriptional repressors. Steady-state kinetic analyses of the selected AlsK and NanK variants demonstrate that their native activities toward d-allose and N-acetyl-d-mannosamine are largely unaffected by the glucokinase-enhancing substitutions. Substrate specificity profiling reveals that the A73G AlsK and L84P NanK variants display systematic improvements in the kcat/Km values for a variety of nonnative carbohydrates. This finding is consistent with an evolutionary process that includes the formation of intermediates possessing relaxed substrate specificities during the initial steps of enzyme functional divergence.
[show abstract][hide abstract] ABSTRACT: Investigations of enzyme action typically focus on elucidating the catalytic roles of hydrogen bonding interactions between polar active-site residues and substrate molecules. Less clear is the importance of non-hydrogen bonding contacts to enzymatic rate accelerations. To investigate the importance of such interactions in a model system, six residues that participate in van der Waals contacts with substrate glucose within the active site of Escherichia coli glucokinase were individually randomized via site-directed mutagenesis. In vivo selection in a glucokinase-deficient bacterium was employed to identify amino acid substitutions that were complicit with enzyme activity. The results suggest that small residues, such as alanine and glycine, are largely immutable, whereas larger amino acids are more tolerant of diverse substitution patterns. Surprisingly, a glucokinase variant that contains glycine in place of six non-hydrogen bonding contacts retains approximately 1% of the wild-type activity. These findings establish non-hydrogen bonding shape determinants as highly appealing targets for widespread substitution during efforts to redesign the catalytic properties of natural enzymes.
Protein Science 10/2007; 16(9):1965-8. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: A review. Topics discussed include: quant. description of enzyme specificity, models of enzyme specificity, advantages and disadvantages of specificity, substrate ambiguity as a mechanism for elaborated metabolic pot., exptl. approaches to detect ambiguity, whole cell mutagenesis and selection, phenotypic screening, overexpression libraries, purifn. of genomic DNA, prepn. of vector DNA, and ligation and transformation of libraries. [on SciFinder(R)]