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PubMed1985

Amino acid metabolism in euryhaline bivalves: regulation of glycine accumulation in ribbed mussel gills.

Glycine levels in isolated ribbed mussel (Modiolus demissus) gill tissue increased slightly and decreased markedly when incubated at high and low salinities, respectively. Low levels of the enzymes involved in the biosynthesis of serine from triose phosphate intermediates, the serine hydroxymethyltransferase, and serine dehydrase were detected in gill tissue homogenates. Experiments using gill tissue incubated with (U-14C)-glycine and (U-14C)-serine indicated interconversion between serine and glycine and transfer of label to alanine, asparate, glutamate, CO2, organic acids, and protein. Glyoxylate was metabolized more slowly than glycine and was probably converted to glycine for catabolism. Studies using (1-14C)-glycine and (2-14C)-glycine with isolated gill tissue and mitochondria indicated that the mitochondrial glycine cleavage enzyme was the major route of glycine catabolism. Metabolic controls activating or inhibiting the glycine cleavage enzyme regulate tissue glycine accumulation and catabolism during hypersalinity or hyposalinity stress.

Amino Acids↗

PubMed1981

Acute myeloblastic leukemia following non-Hodgkin lymphoma in an adolescent. A report of a case with preleukemic syndrome, and review of the literature.

Reports of acute nonlymphoblastic leukemia occurring after successful treatment of Hodgkin and non-Hodgkin lymphoma (NHL) are appearing with increasing frequency. Two years after completion of LSA2-L2 therapy for stage III, poorly differentiated lymphocytic lymphoma, a 16-year-old boy developed a preleukemic state characterized by a refractory macrocytic anemia with excess blasts, dyshematopoiesis, abnormal cluster:colony ratio on in vitro bone marrow culture, and acquired deficiencies of erythrocyte pyruvate kinase, triose phosphate isomerase, and adenylate kinase. Four months later acute myeloblastic leukemia was evident. The RNA index determined by flow cytofluorometry was increased. Four marker chromosomes were found and involved complex translocation of chromosomes 11 and 17 (t11;l17) in 100% of the cells, and chromosomes 4 (t4q;4) in 10% of the cells. A thorough literature search uncovered four other reports of acute nonlymphoblastic leukemia occurring in children treated for NHL and a total of 58 cases in the adult and pediatric age groups. Over 50% of the patients had AML, were mean over 50 years of age, and were treated with radiotherapy and chemotherapy. It is anticipated that additional cases of second malignancies will be reported in this population of patients whose outlook for the curability of the primary malignancy is 75%.

Adolescent↗

PubMed1994

13C NMR study of the generation of C2- and C3-deuterated lactic acid by tumoral pancreatic islet cells exposed to D-[1-13C]-, D-[2-13C]- and D-[6-13C]-glucose in 2H2O.

Tumoral pancreatic islet cells of the RIN5mF line were incubated for 120 min in media prepared in 2H2O and containing D-[1-13C]glucose, D-[2-13C]glucose, and D-[6-13C]glucose. The generation of C2- and C3-deuterated lactic acid was assessed by 13C NMR. The interpretation of experimental results suggests that a) the efficiency of deuteration on the C1 of D-fructose 6-phosphate does not exceed about 47% and 4% in the phosphoglucoisomerase and phosphomannoisomerase reactions, respectively; b) approximately 38% of the molecules of D-glyceraldehyde 3-phosphate generated from D-glucose escape deuteration in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase; and c) about 41% of the molecules of pyruvate generated by glycolysis are immediately converted to lactate, the remaining 59% of pyruvate molecules undergoing first a single or double back-and-forth interconversion with L-alanine. It is proposed that this methodological approach, based on high resolution 13C NMR spectroscopy, may provide novel information on the regulation of back-and-forth interconversion of glycolytic intermediates in intact cells as modulated, for instance, by enzyme-to-enzyme tunneling.

Carbon Isotopes↗

PubMed1995

Lactate dehydrogenase M-subunit deficiencies: clinical features, metabolic background, and genetic heterogeneities.

Lactate dehydrogenase M-subunit deficiency was first reported 1980 as an exertional myoglobinuria. Since then, 6 Japanese and 3 Caucasian families have been reported. Also, typical skin lesions were observed in 1 Japanese patient. Since then, 2 families, one Japanese and one Caucasian, have been found with this typical skin eruption. The metabolic background of exertional myoglobinuria was demonstrated as a result of the impaired reoxidation of NADH produced by glyceraldehyde-3-phosphate-dehydrogenase (G3PD) action. The excess NADH was partly reoxidized by the action of alpha-glycerophosphate dehydrogenase abundant in cytosolic fraction of skeletal muscle. This enzyme reoxidizes excess NADH simultaneously draining out triose phosphate from the glycolytic pathway. Abortive glycolysis results in impaired ATP production followed by rhabdomyolysis. Genomic analysis revealed the heterogeneities of the mutations of this disease.

Adolescent↗

PubMed2002

Changes in phosphate metabolism in thymoma cells suggest mechanisms for resistance to dexamethasone-induced apoptosis. A 31P NMR spectroscopic study of cell extracts.

Treatment of the mouse thymoma-derived WEHI7.2 cell line with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous studies have shown that WEHI7.2 cell variants with an increased antioxidant defense exhibit increased resistance to dexamethasone-induced apoptosis, suggesting that oxidative stress may play a role in glucocorticoid-induced apoptosis. In this work we compared metabolic profiles of WEHI7.2 parental cells with those of WEHI7.2 variants with an increased antioxidant defense or overexpressing bcl-2, to determine whether bolstering the antioxidant defense results in altered metabolic parameters that could translate into increased resistance to dexamethasone-induced apoptosis. WEHI7.2 parental cells and cells overexpressing catalase, thioredoxin or bcl-2, or selected for resistance to 200 micro M H(2)O(2) were cultured in low-glucose DMEM medium supplemented with 10% calf serum, and extracted using chloroform-methanol-water (1:1:1). Metabolites contained in the aqueous and organic phases of the extracts were processed separately and subjected to high-resolution (31)P NMR spectroscopy. In most of the steroid-resistant variants, ATP levels and energetic status were decreased compared with the steroid-sensitive parental cell line, while the concentrations of hexose and triose phosphates were increased. Furthermore, the ratio of choline-containing phospholipids to ethanolamine-containing phospholipids was generally reduced in steroid-resistant cells. Phosphatidylethanolamine and its derivatives contain a higher amount of polyunsaturated fatty acids (PUFA) than the choline-containing analogs, and PUFA are readily oxidized by reactive oxygen species. Therefore, an increased initial amount of phosphatidylethanolamine may increase the 'buffering capacity' of this antioxidant and may thus contribute to the steroid resistance of WEHI7.2 variants.

Animals↗

PubMedDate not supplied

Reflections on the application of 13C-MRS to research on brain metabolism.

The power of (13)C-MRS lies in its unique chemical specificity, enabling detection and quantification of metabolic intermediates which would not be so readily monitored using conventional radiochemical techniques. Examples from animal studies, by examination of tissue extracts from the whole brain, brain slices and cultured cells, include observation of intermediates such as citrate and triose phosphates which have yielded novel information on neuronal/glial relationships. The use of (13)C-labelled acetate as a specific precursor for glial metabolism provided evidence in support of the view that some of the GABA produced in the brain is derived from glial glutamine. Such studies have also provided direct evidence on the contribution of anaplerotic pathways to intermediary metabolism. Analogous studies are now being performed on the human brain, where (13)C-acetate is used to quantitate the overall contribution of glial cells to intermediary metabolism, and use of (13)C-glucose enables direct calculation of rates of flux through the TCA (F(TCA)) and of the glutamate-glutamine cycle (F(CYC)), leading to the conclusion that the rate of glial recycling of glutamate accounts for some 50% of F(TCA). The rate of 0.74 micromol min(-1) g(-1) for F(TCA) is compatible with PET rates of CMRglc of 0.3-0.4 micromol min(-1) g(-1) (since each glucose molecule yields two molecules of pyruvate entering the TCA). Our brain activation studies showed a 60% increase in F(TCA), which is very similar to the increases in CBF and in CMRglc observed in PET activation studies.

Acetates↗

PubMed2005

Proteomic profiling of cholangiocarcinoma cell line treated with pomiferin from Derris malaccensis.

Pomiferin, a prenylated isoflavonoid from Derris malaccensis with strong anti-fungal and anti-oxidant activities, showed cytotoxic activity towards human cholangiocarcinoma cells (HuCCA-1), with IC(50) of 0.9 microg/mL. Pomiferin caused apoptosis, detectable by DNA fragmentation. Two-dimensional PAGE showed increased expression of 12 proteins, namely glucose-regulated protein 75 (grp 75), calcyclin (S100A6), degraded cytokeratin 19, ATP synthase D, ribosomal protein P0, degraded cytokeratin 18 (two spots pI/MW 6.03/29.9 and pI/MW 4.66/21.5), cofilin, annexin A1, triose phosphate isomerase, peroxiredoxin-1, calgizzarin, and profilin. In contrast, cytokeratins (CK) 7, 18 and 19 were down-regulated, and were shown by 1-DE immunodetection to be degraded.

Apoptosis↗

PubMed2006

Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis.

Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere packing did not lead to distinct changes in protein pattern. Applying DPLSR to the 2-DE data enabled the selection of protein spots critical for differentiation between 3 and 6 months frozen storage with 12 months frozen storage. Some of these protein spots have been identified by MS/MS, revealing myosin light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two alpha-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse protein changes in cod muscle proteins during storage has revealed new knowledge on the issue and enables a better understanding of biochemical processes occurring.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

A proteomic analysis of 14-3-3 binding proteins from developing barley grains.

14-3-3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare L.) 14-3-3A was over-expressed, immobilised and used to affinity purify 14-3-3 binding proteins from developing barley grains. Binding was shown to be phosphorylation-dependent. These proteins were fractionated by PAGE and identified by MALDI-TOF MS. In total, 54 14-3-3 binding proteins were identified, 49 of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14-3-3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co-ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14-3-3 in a dosage-dependent manner. The second-largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14-3-3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence.

14-3-3 Proteins↗

PubMed2005

Structures of vertebrate hyaluronidases and their unique enzymatic mechanism of hydrolysis.

Human hyaluronidases (Hyals) are a group of five endo-beta-acetyl-hexosaminidase enzymes, Hyal-1, -2, -3, -4, and PH-20, which degrade hyaluronan using a hydrolytic mechanism of action. Catalysis by these Hyals has been shown to follow a double-displacement scheme. This involves a single Glu residue within the enzyme, the only catalytic residue, as the proton donor (acid). Also involved is a carbonyl group of the hyaluronan (HA) N-acetyl-D-glucosamine as a unique type of nucleophile. Thus the substrate participates in the mechanism of action of its own catalysis. An oxocarbonium ion transition state is postulated, but there is no formation of a covalent enzyme-glycan intermediate, as found in most such reactions. The major domain is catalytic and has a distorted (beta/alpha)8 triose phosphate isomerase (TIM) barrel fold. The C-terminal domain is separated by a peptide linker. Each Hyal has a different C-terminal sequence and structure, the function of which is unknown. These unique C-termini may participate in the additional function(s) associated with these multifunctional enzymes.

Amino Acid Sequence↗

PubMed1991

Energetic approach to the folding of alpha/beta barrels.

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.

Hydrogen Bonding↗

PubMed1993

Glucose phosphorylation in Helicobacter pylori.

Saccharide kinase activities in Helicobacter pylori were investigated by incubating bacterial lysates with ATP and mono- or disaccharides and monitoring directly the appearance of phosphorylated products using 13C or 31P nuclear magnetic resonance spectroscopy. The monosaccharides employed included two trioses, two tetroaldoses, one tetroketose, five aldopentoses, two ketopentoses, five aldohexoses, three ketohexoses, and gluconic and glucuronic acids; the disaccharides studied were maltose, trehalose, cellobiose, sucrose, lactose, gentobiose, and melibiose. D-Glucose was the only sugar phosphorylated among all the carbohydrates examined. The kinase activity of lysates was studied by measuring the rates of formation of glucose 6-phosphate. The substrate specificity, the relatively high KM, and the absence of substrate inhibition suggested that the enzyme is a glucokinase rather than a hexokinase. Most of the glucose kinase activity was observed with the pellet fraction obtained by centrifugation, suggesting an association of the enzyme with the bacterial cell envelope.

Adenosine Triphosphate↗

PubMed1994

The Entner-Doudoroff pathway in Helicobacter pylori.

Evidence for the presence of the enzymes of the Entner-Doudoroff pathway in Helicobacter pylori was obtained using 1H and 31P nuclear magnetic resonance spectroscopy. Bacterial lysates generated 6-phosphogluconate and NADH or NADPH in incubations with glucose-6-phosphate and NAD+ or NADP+, indicating the presence of glucose-6-phosphate dehydrogenase activities. Formation of pyruvate was observed in time courses of incubations of bacterial lysates with 6-phosphogluconate as the only substrate, suggesting the presence of 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase activities. The existence of these enzymes and of triose phosphate isomerase was confirmed by observing the appearance of dihydroxyacetone phosphate in time courses of bacterial lysates incubated with 6-phosphogluconate. Aldolase activity was measured by the production of pyruvate and dihydroxyacetone phosphate in lysates incubated with 2-keto-3-deoxy-6-phosphogluconate as the sole substrate. Dehydrogenase, dehydratase and aldolase activities were observed in several bacterial strains including wild types from fresh isolates. Kinetic parameters were measured for the three activities. The cellular location of the enzymes was investigated by comparing the activities measured in the pellet and supernatant fractions obtained by centrifugation of lysate suspensions. The concentration of compounds causing 50% inhibition of enzyme activity was determined from dose-response curves. The data suggested the presence of two glucose-6-phosphate dehydrogenases linked to NAD+ and NADP+ activities. Using inhibitors differences between the H. pylori and mammalian KDPG aldolases were detected. The presence of these enzyme activities in H. pylori provided evidence for the existence of the Entner-Douderoff pathway in the bacterium.

Fructose-Bisphosphate Aldolase↗

PubMed1994

Metabolic effects and fate of succinic acid methyl esters in rat hepatocytes.

The metabolic fate and metabolic effects of succinic acid methyl esters were examined in rat isolated hepatocytes. Both the monomethyl ester (SAM) and dimethyl ester (SAD) of succinic acid inhibited D-glucose metabolism. Such an inhibition affected, in order of increasing severity, the direct incorporation of D-glucose into glycogen and futile cycling between the hexose and its 6-phosphate ester, the phosphorylation of D-glucose, the generation of triose phosphates from the hexose, and the production of 14C-labeled lactate, pyruvate, and amino acids from D-[U-14C]glucose and its oxidation. The dimethyl ester of [1,4-14C]succinic acid ([1,4-14C]SAD) or [2,3-14C]-succinic acid ([2,3-14C]SAD) was efficiently converted to acidic metabolites. The oxidation of [1,4-14C]SAD largely exceeded that of [2,3-14C]SAD. Inversely the generation of newly formed radioactive D-glucose and glycogen appeared higher in cells exposed to [2,3-14C]SAD, rather than [1,4-14C]SAD. It is proposed that SAM and SAD are suitable nutrients both to cover the energy need of hepatocytes and to act as gluconeogenic precursors.

Amino Acids↗

PubMed1997

Regulatory role of fructose-2,6-bisP on glucose metabolism in frog oocytes: in vivo inhibition of glycogen synthesis.

Glycogen synthesis following glucose microinjection in frog oocytes proceeds preferentially by an indirect pathway involving gluconeogenesis from triose compounds. Because of the known regulatory role of fructose-2,6-bisP on glucose utilization in most vertebrate tissues we coinjected [U-14C]glucose and fructose-2,6-bisP into oocytes and observed a marked inhibition of label incorporation into glycogen, with an I50 value of 2 microM, which is similar to the value measured for the in vitro inhibition of oocyte fructose-1,6-bisphosphatase. Other hexoses-bisP were tested: 2,5-anhydromannitol-1,6-bisP was as effective as inhibitor as fructose-2,6-bisP; glucose-1,6-bisP showed some effect although 50% inhibition was obtained at a concentration 10 times higher than with fructose-2,6-bisP; fructose-1,6-bisP had no effect at all. The inhibition pattern for the in vivo glycogen synthesis by these analogs closely matched the one obtained with partially purified oocyte fructose-1,6-bisphosphatase. The intracellular concentration of fructose-2,6-bisP in unperturbed oocytes was found to be between 0.1 and 0.2 microM. Fructose-6-phosphate,2-kinase levels measured in oocyte homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP microinjected into the oocytes was almost completely degraded, suggesting that fructose-2,6-bisphosphatase is active in vivo. The results presented in this paper indicate that fructose-2,6-bisP plays an important role in the in vivo regulation of glucose utilization in frog-grown oocytes.

Animals↗

PubMed1998

Purification and characterization of cytosolic fructose-1, 6-bisphosphate aldolase from endosperm of germinated castor oil seeds.

Cytosolic fructose-1,6-bisphosphate (FBP) aldolase (ALDc) from the endosperm of 4- to 5-day germinated castor oil seeds (COS) has been purified 83-fold to electrophoretic homogeneity and a final specific activity of 2.5 micromol FBP cleaved/min/mg protein. SDS-PAGE and denaturing isoelectric focusing of the final preparation revealed a single protein-staining band of 40 kDa and pI value 7.2. The native Mr was determined by gel-filtration chromatography and multiangle laser light scattering to be 160-175 kDa, indicating that the enzyme is homotetrameric. The enzyme (a) is a class I aldolase, since EDTA or Mg2+ had no effect on its activity; and (b) was relatively heat stable and had an activation energy of 100 kJ/mol. It exhibited a broad pH-activity optima of 7.2, a relatively high affinity for FBP (Km = 0.16 microM), and a forward:reverse Vmax ratio of 0.77. Rabbit anti-(COS ALDc) antibodies cross-reacted with COS ALDc, but not with the corresponding plastidic isozyme. Time-course studies revealed that (a) the increase in total ALD activity that occurs during COS development and early germination coincides with an increase in ALDc concentration and (b) the latter stages of COS maturation and germination are accompanied by marked reductions in ALD activity and ALDc concentration. The most significant elevation in ALDc concentration occurred over the first 4 days of germination when COS initiates the gluconeogenic conversion of P-enolpyruvate and triose-P, derived from reserve triacylglycerols, into the sucrose required to support early seedling growth.

Amino Acids↗

PubMed2000

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes.

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.

3-Hydroxybutyric Acid↗

PubMed2000

Low-barrier hydrogen bonds and enzymatic catalysis.

Short, strong (low barrier) hydrogen bonds occur when the pK values of the atoms sharing the proton are similar. The overall distance is 2.5 A or less, the deuterium fractionation factor is less than 0.5, the proton NMR chemical shift can approach 20 ppm, and deuterium or tritium substitution causes an up-field change in the chemical shift. Such bonds can have deltaH values of 25 kcal/mol in the gas phase, and at least half that in water or other high-dielectric medium. The strength of the hydrogen bond in an active site drops by approximately 1 kcal/mol for each pH unit mismatch in pKs. When a weak hydrogen bond in the initial enzyme-substrate complex is converted into a low-barrier one by alteration of the pK of the substrate or catalytic group so that the pKs match, the increase in hydrogen bond strength can be used to help catalyze the reaction. A well-established example of this is the reaction catalyzed by serine proteases. The pK of neutral histidine is 14, while that of aspartate is approximately 6. Proton transfer from serine to permit attack on bound substrate produces protonated histidine, with a pK now matching that of aspartate. Studies with trifluoromethyl ketone inhibitors that form tetrahedral adducts show up to five orders of magnitude in binding strength as the result of formation of a low-barrier hydrogen bond between aspartate and histidine. Other enzymes whose mechanisms appear to involve low-barrier hydrogen bonds include liver alcohol dehydrogenase, steroid isomerase, triose-P isomerase, aconitase, citrate synthase, and zinc proteases. It is likely that low-barrier hydrogen bonds form at the transition state of any reaction involving general-acid or general-base catalysis, as at that point the pKs of the catalytic group and reactant will be equal.

Binding Sites↗