Conserved Water Mediated H-bonding Dynamics of Inhibitor,Cofactor, Asp 364 and Asn 303 in Human IMPDH II

Abstract

The IMPDH (Inosine monophosphate dehydrogenase)-II is largely produced in cancer cells. Extensive MD-simulation (2 ns) of the 1B3O, 1NFB, 1NF7, 1LRT, and 1MEW PDB-structures revealed the presence of a conserved water molecule, which is H-bonded and stabilized by the surrounding ribose hydroxyl (O2) of inhibitor, nitrogen (NN) of cofactor, carboxyl oxygen (OD2) and amide nitrogen atoms of the active site Asp 364 and Asn 303 of human. These water-mediated interaction are partially supported in the solvated and X-ray structures. The stereochemistry of the four- centered H-bonds around the conserved water center may be exploited to design a better model inhibitor for IMPDH-II.     

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10?ns simulation of the IMP–NAD⁺ complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD⁺. Three conserved water molecules (W1, W, and W1′) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD⁺) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP–NAD⁺-enzyme complexes and their recognition to NAD⁺, some covalent modification at carboxamide group of di-nucleotide (NAD⁺) has been made by substituting the –CONH₂group by –CONHNH₂ (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.     

Roles of his205, his296, his303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase.

The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56-fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by approximately 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the D-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.     

Studies of the mechanism of phenol hydroxylase: mutants Tyr289Phe, Asp54Asn, and Arg281Met

Three residues in the active site of the flavoprotein phenol hydroxylase (PHHY) were independently changed by site-directed mutagenesis. One of the mutant forms of PHHY, Tyr289Phe, is reduced by NADPH much slower than is the wild-type enzyme, although it has a slightly higher redox potential than the wild-type enzyme. In the structure of the wild-type enzyme, residue Tyr289 is hydrogen-bonded with the FAD when the latter is at the "out" position but has no direct contact with the flavin when it is "in". The oxidative half-reaction of PHHY is not significantly affected by this mutation, contrary to the concept that Tyr289 is a critical residue in the hydroxylation reaction [Enroth, C., Neujahr, H., Schneider, G., and Lindqvist, Y. (1998) Structure 6, 605-617; Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. Tyr289 may help stabilize the FAD in the out conformation where it can be reduced by NADPH. For the Asp54Asn mutant form of PHHY, the initial step of the oxidative half-reaction is significantly slower than for the wild-type enzyme. Asp54Asn utilizes less than 20% of the reduced flavin for hydroxylating the substrate with the remainder forming H(2)O(2). Similar changes are observed when Arg281, a residue between Asp54 and the solvent, is mutated to Met. These two residues are suggested to be part of the active site environment the enzyme provides for the flavin cofactor to function optimally in the oxidative half-reaction. In the construction of the mutant forms of PHHY, it was determined that 11 of the previously reported amino acid residues in the sequence of PHHY were incorrect.     

Role of the Calcium-Binding Residues Asp231, Asp233, and Asp438 in Alpha-Amylase of Bacillus amyloliquefaciens as Revealed by Mutational Analysis

Role of the calcium-binding residues Asp231, Asp233, and Asp438 of Bacillus amyloliquefaciens α-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233, and Asp438 were replaced with Asn, Asn, and Gly to produce the mutants D231N, D233N, and D438G, respectively. The mutant amylases were purified to homogeneity and the purified enzymes was estimated to be approximately 58 kDa. The specific activity for the mutant enzyme D233N was decreased by 84.8%, while D231N and D438G showed a decrease of 6.3% and 3.5% to that of the wild-type enzyme, respectively. No significant changes in the K _m value, thermo-stability, optimum temperature, and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (K _cat/K _m) and thermo-stability at 60°C. The ranges of optimum temperature and optimum pH for D233N were also reduced to about 10°C and 3–4 units, respectively.     

A conserved Asn in TM7 of the thyrotropin receptor is a common requirement for activation by both mutations and its natural agonist

Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies.     

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+

Aldo-keto reductases of family 2 employ single site replacement Lys-->Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3A, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.     

Three-dimensional structure prediction of bovine AP lyase, BAP1: prediction of interaction with DNA and alterations as a result of Arg176-->Ala, Asp282-->Ala, and His308-->Asn mutations

BAP1 is an apurinic/apyrimidinic lyase (AP lyase) that plays an important role in the repair of DNA damage. The present study deals with the prediction of the 3D structure of bovine AP lyase based on its sequence homology with human AP lyase. The predicted 3D model of bovine AP1 shows remarkable similarity with human endonuclease in the overall 3D fold. However, significant differences in the model and the X-ray structure were located at some of the important sites. We have analyzed the active center of the enzyme and other sites that are involved in DNA repair. A number of amino acids bind the bases located in the major/minor grooves of DNA. An insertion of Arg176 in the major groove and Met270 in the minor groove caps the DNA bound enzyme's active site, stabilizing the extra helical AP site conformation and effectively locking the protein onto the AP-DNA. Three BAP1 mutants were also modeled and analyzed as regards the changes in the structure. Substitution of Arg176-->Ala leads to the loss of DNA binding whereas mutation of Asp282-->Ala and His308-->Asn leads to a decrease in the enzymatic activity.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the charge interaction between Arg(70) and Asp(120) in the Tn10-encoded metal-tetracycline/H(+) antiporter of Escherichia coli

We reported that the positive charge of Arg(70) is mandatory for tetracycline transport activity of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Arg(70) may function through a charge-pairing with a negatively charged residue in close proximity. Therefore, we mutated Asp(66) and Asp(120), which are only two negatively charged residues located close to Arg(70) in putative secondary structure of TetA(B) and highly conserved throughout transporters of the major facilitator superfamily. Site-directed mutagenesis studies revealed that Asp(66) is essential, but Asp(120) is important for TetA(B) function. Surprisingly, when Asp(120) was replaced by a neutral residue, the R70A mutant recovered tetracycline resistance and transport activity. There was no such effect in the Asp(66) mutation. The charge-exchanged mutant, R70D/D120R, also showed significant drug resistance and transport activity (about 50% of the wild type), although the R70D mutant had absolutely no activity, and the D120R mutant retained very low activity (about 10% of the wild type). Both the R70C and D120C mutants were inactivated by N-ethylmaleimide. Mercuric ion (Hg(2+)), which gives a positive charge to a SH group of a Cys residue through mercaptide formation, had an opposite effect on the R70C and D120C mutants. The activity of the R70C mutant was stimulated by Hg(2+); however, on the contrary, the D120C mutant was partially inhibited. On the other hand, the R70C/D120C double mutant was almost completely inactivated by Hg(2+), probably because the side chains at positions 70 and 120 are bridged with Hg(2+). The close proximity of positions 70 and 120 were confirmed by disulfide cross-linking formation of the R70C/D120C double mutant when it was oxidized by copper-(1,10-phenanthroline). These results indicate that the positive charge of Arg(70) requires the negative charge of Asp(120) for neutralization, probably for properly positioning transmembrane segments in the membrane.     

Functional consequences of alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the interfaces between domain P, M3, and L6-7 of sarcoplasmic reticulum Ca2+-ATPase. Roles in Ca2+ interaction and phosphoenzyme processing

Point mutants with alterations to amino acid residues Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed by transient kinetic measurements. In the Ca(2+)-ATPase crystal structures, most of these residues participate in a hydrogen-bonding network between the phosphorylation domain (domain P), the third transmembrane helix (M3), and the cytoplasmic loop connecting the sixth and the seventh transmembrane helices (L6-7). In several of the mutants, a pronounced phosphorylation "overshoot" was observed upon reaction of the Ca(2+)-bound enzyme with ATP, because of accumulation of dephosphoenzyme at steady state. Mutations of Glu(340) and its partners, Thr(247) and Arg(822), in the bonding network markedly slowed the Ca(2+) binding transition (E2 --> E1 --> Ca(2)E1) as well as Ca(2+) dissociation from Ca(2+) site II back toward the cytosol but did not affect the apparent affinity for vanadate. These mutations may have caused a slowing, in both directions, of the conformational change associated directly with Ca(2+) interaction at Ca(2+) site II. Because mutation of Asp(813) inhibited the Ca(2+) binding transition, but not Ca(2+) dissociation, and increased the apparent affinity for vanadate, the effect on the Ca(2+) binding transition seems in this case to be exerted by slowing the E2 --> E1 conformational change. Because the rate was not significantly enhanced by a 10-fold increase of the Ca(2+) concentration, the slowing is not the consequence of reduced affinity of any pre-binding site for Ca(2+). Furthermore, the mutations interfered in specific ways with the phosphoenzyme processing steps of the transport cycle; the transition from ADP-sensitive phosphoenzyme to ADP-insensitive phosphoenzyme (Ca(2)E1P --> E2P) was accelerated by mutations perturbing the interactions mediated by Glu(340) and Asp(813) and inhibited by mutation of Pro(248), and mutations of Thr(247) induced charge-specific changes of the rate of dephosphorylation of E2P.     

Role of the conserved acidic residue Asp21 in the structure of phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and nuclear magnetic resonance studies

To elucidate a role of the Src homology 3 (SH3)-conserved acidic residue Asp21 of the phosphatidylinositol 3-kinase (PI3K) SH3 domain, structural changes induced by the D21N mutation (Asp21 --> Asn) were examined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. In the previous study, we demonstrated that environmental alterations occurred at the side chains of Trp55 and some Tyr residues from the comparison of the near-UV CD spectra of the PI3K SH3 domain with or without a D21N mutation [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr14 and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue with or without a D21N mutation. The (1)H and (15)N resonance assignments of the PI3K SH3 domain and its D21N mutant revealed that significant chemical shift changes occurred to the aromatic side-chain protons of Trp55 and Tyr14 upon the D21N mutation. All these aromatic residues are implicated in ligand recognition. In addition, the NMR analysis showed that the backbone conformations of Lys15-Asp23, Gly54-Trp55, Asn57-Gly58, and Gly67-Pro70 were affected by the D21N mutation. Furthermore, the (15)N[(1)H] nuclear Overhauser effect values of Tyr14, Glu19, and Glu20 were remarkably changed by the mutation. These results show that the D21N mutation causes structural deformation of more than half of the ligand binding cleft of the domain and provide evidence that Asp21 plays an important role in forming a well-ordered ligand binding cleft in cooperation with the RT loop (Lys15-Glu20).     

Insight towards the conserved water mediated recognition of catalytic and structural Zn+2 ions in human Matrix Metalloproteinase-8 enzyme: A study by MD-simulation methods

Human matrix metalloproteinase-8 (hMMP-8) plays a important role in the progression of colorectal cancer, metastasis, multiplesclerosis and rheumetoid arthritis. Extensive MD-simulation of the PDB and solvated structures of hMMP-8 has revealed thepresence of few conserved water molecules around the catalytic and structural zinc (ZnC and ZnS) ions. The coordination of twoconserved water molecules (W and WS) to ZnS and the H-bonding interaction of WS to S151 have indicated the plausible involvementof that metal ion in the catalytic process. Beside this the coupling of ZnC and ZnS metal ions (ZnC – W_H (W₁)…..W₂ ….H₁₆₂ - ZnS)through two conserved hydrophilic centers (occupied by water molecules) may also provide some rational on the recognition oftwo zinc ions which were separated by ~13 Å in their X-ray structures. This unique recognition of both the Zn⁺² ions in the enzymethrough conserved water molecules may be implemented/ exploited for the design of antiproteolytic agent using water mimicdrug design protocol.     

Effects of overexpression of NAPRTase,NAMNAT, and NAD synthetase in the NAD(H) biosynthetic pathways on the NAD(H) pool,NADH/NAD+ ratio,and succinic acid production with different carbon sources by metabolically engineered Escherichia coli

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose anaerobically due to the inability to regenerate NAD⁺. To regulate NAD(H) pool size and NADH/NAD⁺ ratio, overexpression of the enzymes in the NAD(H) biosynthetic pathways in BA002 was investigated. The results clearly demonstrate that the increased NAD(H) pool size and the decreased NADH/NAD⁺ ratio improved the glucose consumption and cell growth, which improved succinic acid production. When the pncB and the nadD genes were co-overexpressed in CA102, the ratio of NADH/NAD⁺ was decreased from 0.60 to 0.12, and the concentration of NAD(H) was the highest among that of all the strains. Moreover, the dry cell weight (DCW), glucose consumption, and the concentration of succinic acid in CA102 were also the highest. Based on the sufficient NAD⁺ supply after gene modification in the NAD(H) biosynthetic pathways, reductive carbon sources with different amounts of NADH can further change the distribution of metabolites. When sorbitol was used as a carbon source in CA102, the byproducts were lower than those of glucose fermentation, and the yield of succinic acid was increased.     

Synergistic interactions of multiple mutations on catalysis during the hydroxylation reaction of p-hydroxybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp, and Tyr385Phe mutants reconstituted with 8-Cl-flavin

The oxygen transfer to p-hydroxybenzoate catalyzed by p-hydroxybenzoate hydroxylase (PHBH) has been shown to occur via a C4a-hydroperoxide of the flavin. Two factors are likely to be important in facilitating the transfer of oxygen from the C4a-hydroperoxide to the substrate. (a) The positive electrostatic potential of the active site partially stabilizes the negative charge centered on the oxygen of the flavin-C4a-alkoxide leaving group during the transition state [Ortiz-Maldonado, M., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 8124-8137]. (b) The hydrogen-bonding network ionizes the substrate to promote its nucleophilic attack on the electrophilic C4a-hydroperoxide intermediate [Entsch, B., Palfey, B. A., Ballou, D. P., and Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. This ionization is also aided by the positive electrostatic potential of the active site [Moran, G. R., Entsch, B., Palfey, B. A., and Ballou, D. P. (1997) Biochemistry 36, 7548-7556]. Substituents on the flavin can specifically affect the stability of the alkoxide leaving-group, whereas changes to specific enzyme residues can affect the charge in the active site and the hydrogen-bonding network. We have used wild-type (WT) PHBH and several mutant forms, all with normal FAD and with 8-Cl-FAD substituted for FAD, to assess the relative contributions of the two effects. Lys297Met and Asn300Asp have decreased positive charge in the active site, and these variants engender approximately 35-fold slower hydroxylation rates than the WT enzyme. Substitution of 8-Cl-FAD in these mutant forms gives approximately 1.8-fold increases in hydroxylation rates, compared with a > or =4.8-fold increase for WT with this flavin. The hydroxylation catalyzed by Tyr385Phe, a mutant enzyme form with a disrupted hydrogen-bonding network that compromises the ionization of the substrate without changing the positive charge of the active site, is stimulated 1.5-fold by substituting the enzyme with 8-Cl-FAD. The substrate, tetrafluoro-p-hydroxybenzoate, is fully ionized in WT PHBH, but this phenolate is a poor nucleophile because of the electron-withdrawing effects of the fluorine substituents. With tetrafluoro-p-hydroxybenzoate as the substrate, substitution of FAD with 8-Cl-FAD in the WT enzyme stabilizes the leaving alkoxide and leads to a 2.3-fold increase in the hydroxylation rate compared to that with FAD. Either the use of substrates that do not communicate with the proton network or the mutation of amino acid residues that perturb this interaction may prevent a necessary conformational change that allows proper orientation between reactants during the hydroxylation reaction or permits the essential protonation of the initially formed nascent flavin-C4a-peroxide anion. Thus, both activation of substrate by the proton network and stabilization of the leaving alkoxide appear to be important for oxygen transfer catalyzed by PHBH. The full effect of the substituents on the flavin (4.8-fold) can only be realized when the optimal transition state can be achieved, and this optimal state is not fully realized with the mutant forms.     

Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-(32)P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-(14)C]NAD individually upon UV irradiation, but most (14)C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-(14)C]NAD suggested that Arg 101 is not absolutely required for NAD binding.     

Hb J-Singa (alpha-78 Asn leads to Asp), a newly discovered hemoglobin variant with the same amino acid substitution as one of the two present in Hb J-Singapore (alpha-78 Asn leads to, alpha-79 Ala leads to Gly)

A new fast-moving alpha-chain Hb variant with an Asn leads to Asp substitution at position alpha-78 was found in a French-Acadian family living in Eastern Canada. The identical substitution was reported in Hb J-Singapore, which also had an additional Ala leads to Gly substitution at position alpha-79. The new variant, which did not result in any clinical symptoms, was named accordingly, Hb J-Singa.     

Role of obesity and hyperinsulinemia in the insulin resistance of obese subjects with the clinical triad of polycystic ovaries, hirsutism and acanthosis nigricans

The insulin resistance of 4 nonobese and 8 obese patients with polycystic ovaries, hirsutism and benign acanthosis nigricans, and of 6 'obese normal' apart from obesity and 10 normal female subjects was evaluated by means of an intravenous insulin tolerance test and by measuring basal and insulin responses to an oral glucose load. The patients with polycystic ovaries, hirsutism and acanthosis had a decreased hypoglycemic response to exogenous insulin. The subjects with polycystic ovaries presented a significantly greater mean glucose response area for the same or greater mean insulin response area than the obese or nonobese normal subjects. The insulin resistance in the patient with polycystic ovaries, hirsutism and acanthosis nigricans could not be exclusively ascribed to a reduced receptor number, but also appeared to be due to a simultaneous postbinding defect probably related to the high insulin levels in patients with polycystic ovaries be they obese or not. The elevated plasma androgens and the presence of acanthosis found in these patients are likely also related to the hyperinsulinemia. To evaluate the influence of obesity, obese and nonobese patients with acanthosis nigricans and polycystic ovaries were compared. Higher insulin levels were found in the thin subjects, which could explain their greater insulin resistance and more severe hyperandrogenism. The comparison between obese patients with and those without acanthosis nigricans and polycystic ovaries suggested that, despite similar insulin levels, the greater known duration of obesity (probably also of the hyperinsulinemia) of the former was a possible explanation for their more intense insulin resistance and higher testosterone levels.     

Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae

beta-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (beta-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nepsilon stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nepsilon of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.