Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.     

Loop diuretics act directly on adenylate cyclase in rat renal tubular basolateral membranes

We have reported previously that loop diuretics, especially azosemide and ethacrynic acid, may act not only on the AVP receptor site, but also on the post-AVP receptor site in rat renal tubular basolateral membranes. The purpose of this study was to examine whether loop diuretics (furosemide, azosemide, ethacrynic acid) affect the post-AVP receptor components, using GTP-gamma S, forskolin and cholera toxin as tools acting distal to the receptor. Adenylate cyclase activity stimulated by 10(-9)M AVP was inhibited more potently by azosemide and ethacrynic acid than by furosemide at the concentration of 10(-3) M. Azosemide and ethacrynic acid at concentrations above 10(-4) M also significantly decreased the enzyme activity that was stimulated by 10(-7) M GTP-gamma S and 10(-5)M forskolin, while significant inhibition by furosemide was observed only at 10(-3)M. In addition, the inhibitory effect of these loop diuretics on cholera toxin-stimulated enzyme activity was almost similar to the results observed in AVP-, GTP-gamma S- or forskolin-stimulated the enzyme activity. From these results, we conclude that loop diuretics, especially azosemide and ethacrynic acid, directly affect adenylate cyclase in part as well as the AVP receptor site.     

Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase

We have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 --> Leu, Asp130 --> Gln, Glu131 --> Gln, and Lys134 --> Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 x 10(9) M(-1) s(-1), at pH 7 and mu = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.     

Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition

The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4. 1.1.31) has been determined by x-ray diffraction methods at 2.8-A resolution by using Escherichia coli PEPC complexed with L-aspartate, an allosteric inhibitor of all known PEPCs. The four subunits are arranged in a "dimer-of-dimers" form with respect to subunit contact, resulting in an overall square arrangement. The contents of alpha-helices and beta-strands are 65% and 5%, respectively. All of the eight beta-strands, which are widely dispersed in the primary structure, participate in the formation of a single beta-barrel. Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers. The location of the catalytic site is likely to be near the C-terminal side of the beta-barrel. The binding site for L-aspartate is located about 20 A away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding. The participation of Arg-587 is unexpected, because it is known to be catalytically essential. Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC, L-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site. There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure. The importance of this loop in catalytic activity was also shown. Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by L-aspartate.     

Substrate specificities of alpha-L-arabinofuranosidases produced by two species of Aspergillus niger

Precise substrate specificities of alpha-L-arabinofuranosidases from Aspergillus niger 5-16 and Aspergillus niger (Megazyme) were investigated. Both enzymes hydrolyzed arabinan and debranched-arabinan at almost the same rate. The alpha-L-Arabinofuranosidase from A. niger (Megazyme) preferentially released arabinosyl side-chains of arabinan. The enzyme tore off both arabinoses attached to O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1--> 4)-D-xylopyranose and O-beta-D-xylopyranosyl-(1-->4)-[O-alpha-L- arabinofuranosyl-(1-->3)]-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose, but did not tear off xylosyl-arabinose from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L-arabinofuranosyl-(1-->3) -O-beta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyranosyl-(1-->4)-D- xylopyranose. The enzyme from A. niger (Megazyme) hydrolyzed methyl 2-O-, methyl 3-O- and methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranosides to arabinose and methyl alpha-L-arabinofuranoside in the order of (1-->5)->(1-->2)->(1-->3)-linkages. On the other hand, alpha-L-arabinofuranosidase from A. niger 5-16 successively liberated the arabinose of arabinan from non-reducing terminals. The enzyme hydrolyzed in the order of (1-->2- > (1-->3)- > (1-->5)-linkages. Both of the enzymes hydrolyzed the (1-->3)-linkage more than the (1-->5)-linkage of methyl 3,5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside.     

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 --> Lys mutation.     

Fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species occurs near Gly-329

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the illuminated lysates of wheat (Triticum aestivum L.) chloroplasts is broken down by reactive oxygen radicals into 37- and 16-kDa polypeptides. Analysis of the terminal amino acid residues of both fragments revealed that the C terminus of the 37-kDa fragment was Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329, which links the two fragments, was missing, suggesting that the fragmentation of the LSU in the lysates driven by oxygen-free radicals occurs at Gly-329. Purified rubisco, exposed to a hydroxyl radical-generating system, was also cleaved at the same site of the LSU. The cleavage site was positioned at the N-terminal end of the flexible loop (loop 6) within the beta/alpha-barrel domain, constituting the catalytic site of rubisco. The binding of a reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of rubisco completely protected the enzyme from the fragmentation. The fragmentation was differentially affected by CO2, Mg2+, ribulose 1, 5-bisphosphate, or 2-carboxyarabinitol 1,5-bisphosphate. All these results indicate that the conformation of the catalytic site of the enzyme is involved as an important factor determining the breakdown of rubisco by reactive oxygen species. Reactive oxygen species generated at its catalytic site by a Fenton-type reaction may trigger the site-specific degradation of the LSU in the lysates of chloroplasts in the light.     

Residue lysine-34 in GroES modulates allosteric transitions in GroEL

The conserved residue Lys-34 in GroES was replaced by alanine and glutamic acid using site-directed mutagenesis. This residue is near the carboxy terminus of the mobile loop in GroES (residues 17-32) which becomes immobilized upon formation of the GroEL/GroES complex [Landry et al. (1993) Nature 364, 255-258]. Both charge neutralization (Lys-34-->Ala) and charge reversal (Lys-34-->Glu) at this position have little effect on the binding constant of GroES to GroEL, but they increase the enhancement by GroES of cooperativity in ATP hydrolysis by GroEL. This is reflected by a change in the Hill coefficient (at 10 mM K+) from 4.10 (+/- 0.22) in the presence of wild-type GroES to 5.17 (+/- 0.24) and 4.46 (+/- 0.14) in the presence of the GroES mutants Lys-34-->Ala and Lys-34-->Glu, respectively. The results are interpreted using the Monod-Wyman-Changeux (MWC) model for cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. They suggest that Lys-34 in GroES modulates the allosteric transition in GroEL by stabilizing a relaxed (R)-like state.     

Chemoenzymatic synthesis of a 3IV,6III-disulfated Lewis(x) pentasaccharide, a candidate ligand for human L-selectin

The disulfated pentasaccharide 3-O-SO3(-)-beta-D-Galp-(1-->4)-[alpha-L-Fucp-(1-->3)]-6-O-SO3(-)- beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-Glcp was prepared according to a chemoenzymatic approach, starting from 4-methoxybenzyl O-(4-O-acetyl-2,6-di-O-benzyl-beta- D-galactopyranosyl)-(1-->4)-O-2,3,6-tri-O-benzyl-beta-D-glucopyranoside, obtained in six steps from hepta-O-acetyl lactosyl bromide. Coupling of this lactose derivative with O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl) trichloracetimidate afforded, after dephthaloylation and re-N-acetylation, 4-methoxybenzyl O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1-->3)-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-- >4)- O-2,3,6-tri-O-benzyl-beta-D-glucopyranoside. Regioselective sulfation at the primary position of the glucosamine residue was then successfully achieved and the benzyl groups were removed. Enzymatic galactosylation of 4-methoxybenzyl O-(2-acetamido-2-deoxy-6-O-sulfo-beta-D- glucopyranosyl)-(1-->3)-O-beta-D-galactopyranosyl-(1-->4)-O-beta-D- glucopyranoside sodium salt, and subsequent regioselective sulfation at position 3 of the outer galactose residue through the stannylene procedure, led then to 4-methoxybenzyl O-(3-sulfo-beta-D- galactopyranosyl)-(1-->4)-O-(2-acetamido-2-deoxy-6-sulfo-beta-D- glucopyranosyl)-(1-->3)-O-beta-D-galactopyranosyl)-(1-->4)-O-beta-D- glucopyranoside disodium salt, which was finally fucosylated using human milk alpha-(1-->3/4)-fucosyltransferase affording, after anomeric deprotection, the target pentasaccharide.     

Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl(alpha 1-->6)-D-myo-inositol, an essential fragment of GPI anchors

Numerous proteins on the external surface of the plasma membrane are anchored by glycosylated derivatives of phosphatidylinositol (GPI), rather than by hydrophobic amino acids embedded in the phospholipid bilayer. These GPI anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) to release a water-soluble protein with an exposed glycosylinositol moiety and diacylglycerol, which remains in the membrane. We have previously determined the crystal structure of Bacillus cereus PI-PLC, the enzyme which is widely used to release GPI-anchored proteins from membranes, as free enzyme and also in complex with myo-inositol [Heinz, D.W., Ryan, M. Bullock, T.L., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863]. Here we report the refined 2.2 A crystal structure of this enzyme complexed with a segment of the core of all GPI anchors, glucosaminyl(alpha 1-->6)-D-myo-inositol [GlcN-(alpha 1-->6)Ins ]. The myo-inositol moiety of GlcN(alpha 1-->6)Ins is well-defined and occupies essentially the same position in the active site as does free myo-inositol, which provides convincing evidence that the enzyme utilizes the same catalytic mechanism for cleavage of PI and GPI anchors. The myo-inositol moiety makes several specific hydrogen bonding interactions with active site residues. In contrast, the glucosamine moiety lies exposed to solvent at the entrance of the active site with minimal specific protein contacts. The glucosamine moiety is also less well-defined, suggesting enhanced conformational flexibility. On the basis of the positioning of GlcN(alpha 1-->6)Ins in the active site, it is predicted that the remainder of the GPI-glycan makes little or no specific interactions with B. cereus PI-PLC. This explains why B. cereus PI-PLC can cleave GPI anchors having variable glycan structures.     

Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum

An extracellular beta-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0-6.0 and at 60 degrees C. It hydrolysed 1-->4-linked aryl-beta-glucosides and 1-->4-linked, 1-->3-linked and 1-->6-linked beta-glucosides. The apparent Km and kcat values for p-nitrophenyl beta-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 microM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, beta-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.     

Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity

Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.     

Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 2. Characterization of E-selectin binding fractions, and structural requirements for physiological binding to E-selectin

E-selectin binding gangliosides were isolated from myelogenous leukemia HL60 cells, and the E-selectin binding pattern was compared with that of human neutrophils as described in the preceding paper in this issue. The binding fractions were identified as monosialogangliosides having a series of unbranched polylactosamine cores. Structures of fractions 12-3, 13-1, 13-2, and 14, which showed clear binding to E-selectin under the conditions described in the preceding paper, were characterized by functional group analysis by application of monoclonal antibodies, 1H-NMR, FAB-MS, and electrospray mass spectrometry with collision-induced dissociation of permethylated fractions. Fractions 12-3, 13-1, and 13-2 were characterized by the presence of a major ganglioside with the following structure: NeuAc alpha 2-->3Gal beta 1-->4 GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer. Fractions 12-3 and 13-2 contained, in addition, small quantities (10-15%) of extended SLex with internally fucosylated structures: NeuAc alpha 2-->3 Gal beta 1-->4-(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4 (+/- Fuc alpha 1-->3)GlcNA c beta 1-->3 Gal beta beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->Glc Beta Cer. Fraction 13-1, showing stronger E-selectin binding activity than 12-3 and 13-2, contained only a trace quantity (< 1%) of SLex. Fraction 14, which also showed clear binding to E-selectin, was characterized by the presence of the following structures, in addition to two internally monofucosylated structures (XX and XXI, Table 2, text): NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer; andNeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4 (Fuc alpha 1--3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1--4Glc beta Cer. SLex determinant was completely absent. Thus, the E-selectin binding epitope in HL60 cells is carried by unbranched terminally alpha 2-->3 sialylated polylactosamine having at least 10 monosaccharide units (4 N-acetyllactosamine units) with internal multiple fucosylation at GlcNAc. These structures are hereby collectively called "myeloglycan". Monosialogangliosides from normal human neutrophils showed an essentially identical pattern of gangliosides with selectin binding property. Myeloglycan, rather than SLex, provides a major physiological epitope in E-selectin-dependent binding of leukocytes and HL60 cells.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Hydration and stability of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles

The effect of sulfatide on membrane hydration of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles (SUVs) was investigated using steady-state and time-resolved fluorescence spectroscopy. The degree of hydration in the headgroup region of the bilayer lipids was assessed with the fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine along with the ratio of its fluorescence intensities measured in samples prepared either in D2O- or in H2O-based buffers. Similarly, hydration of acyl chains near the headgroup region and that close to the bilayer center were studied using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3, 5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine as probes. Increasing sulfatide concentration up to 30 mol% resulted in an increase in surface hydration and a decrease in interchain hydration. These were correlated with an increase in bilayer stability of the DOPE/sulfatide SUVs. Moreover, variation of pH was found to affect the hydration and stability of the bilayer vesicles. No further change in headgroup hydration and interchain hydration near the bilayer center was observed at sulfatide concentrations >/=30 mol%. At such high sulfatide concentrations, bilayer hydration and stability were no longer pH-sensitive. The effects of sulfatide on hydration and stability of DOPE bilayer vesicles are discussed by taking into account the electrostatic and geometrical properties of the sulfated galactosyl headgroups.     

Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by x-ray crystallography

Protein-tyrosine phosphatases (PTPs) are signal transduction enzymes that catalyze the dephosphorylation of phosphotyrosine residues via the formation of a transient cysteinyl-phosphate intermediate. The mechanism of hydrolysis of this intermediate has been examined by generating a Gln-262 --> Ala mutant of PTP1B, which allows the accumulation and trapping of the intermediate within a PTP1B crystal. The structure of the intermediate at 2.5-A resolution reveals that a conformationally flexible loop (the WPD loop) is closed over the entrance to the catalytic site, sequestering the phosphocysteine intermediate and catalytic site water molecules and preventing nonspecific phosphoryltransfer reactions to extraneous phosphoryl acceptors. One of the catalytic site water molecules, the likely nucleophile, forms a hydrogen bond to the putative catalytic base, Asp-181. In the wild-type enzyme, the nucleophilic water molecule would be coordinated by the side chain of Gln-262. In combination with our previous structural data, we can now visualize each of the reaction steps of the PTP catalytic pathway. The hydrolysis of the cysteinyl-phosphate intermediate of PTPs is reminiscent of GTP hydrolysis by the GTPases, in that both families of enzymes utilize an invariant Gln residue to coordinate the attacking nucleophilic water molecule.     

Mechanism of aldehyde oxidation catalyzed by horse liver alcohol dehydrogenase

The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed by horse liver alcohol dehydrogenase (HLADH) has been investigated using the HLADH structure at 2.1 A resolution with NAD+ and pentafluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations with HLADH were obtained by a best-fit superimposition of benzaldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II)ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(II) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initio-derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21,40-56]. Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MD1) enzyme with NAD+, benzaldehyde, and zinc-ligated HO-in the active site; (MD2) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxygen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempirical PM3 calculations. Using PM3, model systems were calculated of ground states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc-bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resided at a distance of 2.8-5.5 A from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 transition state for attack of the Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, which has an O...C distance of 1.877 A. HLADH catalysis of the aldehyde hydration would require very little motion aside from that in the ground state. Two simulations of benzaldehyde hydrate ligated to zinc (MD2 and MD3) both showed close approach of the aldehyde hydrate hydrogen to NAD+C4, varying from 2.3 to 3.3 A, seemingly favorable for the hydride transfer reaction. The MD2 configuration does not allow proton shuttling. On the other hand, when the pro-S oxygen is ligated to zinc (MD3), the proton on the pro-R oxygen averages 2.09 A from the hydroxyl oxygen of Ser48 such that initiation of shuttling of protons via Ser48 to the ribose 2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen is sterically favorable. PM3 calculations suggest that this proton shuttle represents a stepwise reaction which occurs subsequent to hydride transfer. The PM3 transition state for hydride transfer based on the MD3 configuration has the transferring hydride 1.476 A from C4 of NAD+ and 1.433 A from the aldehyde alpha-carbon.     

Site-directed mutagenesis clarifies the substrate position within the three-dimensional model of the active site of herpes simplex virus type-1 thymidine kinase

Site-directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type-1 thymidine kinase (HSV 1 TK) obtained by homology modelling. For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir-insensitive bovine herpes virus type-1 thymidine kinase (BHV 1 TK). Wild-type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity. While both mutants had the same Km value for thymidine as the recombinant wild-type enzyme (0.2 microM), Vmax was decreased to 20-25% of the original wild-type value. The recombinant wild-type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 microM. Both mutants were able to phosphorylate aciclovir to about the same extent as the wild-type enzyme. These findings suggest that neither D215 nor K317 are directly involved in substrate binding. Therefore, a rearrangement of the 3D model is suggested, concerning the assignment of the substrate-binding site and co-substrate-binding site at the right and left side of the phosphate-binding loop, respectively.     

Structural requirements of synthetic oligosaccharides to bind monoclonal antibodies against Chlamydia lipopolysaccharide

Monoclonal antibodies were generated against a synthetic glycoconjugate containing the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo (Kdo, 3-deoxy-D-manno-2-octulopyranosonic acid) which represents the genus-specific epitope of the lipopolysaccharide from the obligatory intracellular human pathogen Chlamydia. Antibodies of all immunoglobulin G isotypes were obtained and characterized by enzyme immunobinding and inhibition assays using the immunizing antigen as well as chemically synthesized derivatives of the Kdo trisaccharide. The latter contained (1) one of the three residues in beta- instead of alpha-linkage, (2) a Kdo residue the carboxyl group of which had been reduced to a CH2OH group (Kdo(C1-red)), or (3) changing the linkage of the terminal Kdo from 2-->8 to 2-->4. Only one compound, namely, alpha-Kdo-(2-->8)-alpha-Kdo(C1-red)-(2-->4)-alpha-Kdo exhibited binding to and inhibition of Kdo trisaccharide-specific antibodies, whereas all other compounds were not active. Structural and conformational investigations using NMR spectroscopy at high field on the allyl glycosides of the oligosaccharides 6-12 confirmed the conformational similarities between those structures 4, 5, and 10 which were able to bind to the antibodies investigated.     

High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein

Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain.